Human ESAT-6 FOR RESEARCH USE ONLY Assay range:2pg/ml-48pg/ml 96 determinationsPurposeThis kit allows for the determination of ESAT-6 concentrations in Human serum, cell culture supernates and other biological fluids Principle of the assayThe kit assay Human ESAT-6 level in the sample,use Purified Human ESAT-6 antibody to coat microtiter plate wells, make solid-phase antibody, then add ESAT-6 to wells, Combined ESAT-6 antibody which With HRP labeled goat anti-Human become antibody - antigen - enzyme-antibody complex, after washing Completely, Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of Human ESAT-6 in the samples is then determined by comparing the O.D. of the samples to the standard curve.Materials provided with the kit1 wash solution 20ml×1bottle 7 Stopp Solution 6ml×1 bottle 2 HRP-Conjugate reagent 6ml×1 bottle 8 Standard(960ng/L) 0.5ml×1 bottle 3 Microelisa stripplate 12well×8strips 9 Standard diluent 1.5ml×1bottle 4 Sample diluent 6ml×1 bottle 10 Instruction 1 5 Chromogen Solution A 6ml×1 bottle 11 Closure plate membrane 2 6 Chromogen Solution B 6ml×1 bottle 12 Sealed bags 1 Specimen requirements1. extract as soon as possible after Specimen collection,and according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can’t, specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles.2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.Assay procedure1. Dilute and add sample:Dilute Original density Standard as follow table:480ng/L 5 Standard 150μl Original density Standard+150μl Standard diluent 240ng/L 4 Standard 150μl 5 Standard+150μl Standard diluent 120ng/L 3 Standard 150μl 4 Standard+150μl Standard diluent 60ng/L 2 Standard 150μl 3 Standard +150μl Standard diluent 30ng/L 1 Standard 150μl 2 Standard +150μl Standard diluent 2.add sample:Set blank wells separately (blank comparison wells don’t add sample and HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution 40μl to testing sample well, then add testing sample 10μl (sample final dilution is 5-fold), add sample to wells , don’t touch the well wall as far as possible, and Gently mix.3.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37℃.4.Configurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.5.washing:Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.6.add enzyme:Add HRP-Conjugate reagent 50μl to each well, except blank well. 7.incubate:Operation with 3.8.washing:Operation with 5.9.color:Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 37℃10.Stop the reaction:Add Stop Solution50μl to each well, Stop the reaction(the blue color change to yellow color).11.assay:take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min.Steps descriptionStandard, Sample diluent Add Standard, Sample diluent, incubate for 30 min at 37℃. Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37℃. Wash 5 times,Add Chromogen Solution A and B, incubate for 30 min at 37℃. Add Stopp Solution Read absorbance at 450nm within 15 min calculate CalculateTake the standard density as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find out the corresponding density according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation of the standard curve with the standard density and the OD value ,with the sample OD value in the equation, calculate the sample density, multiplied by the dilution factor, the result is the sample actual density.Important notes1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.2. washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result.3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. add sample within 5 min, if the number of sample is much , recommend to use Volley .4. if the testing material content is excessively higher (The sample OD is bigger than the first standard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilution factor.(×n×5).5. Closure plate membrane only limits the disposable use, to avoid cross-contamination.6. The substrate evade the light preservation.7. Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.8. All samples, washing buffer and each kind of reject should according to infective material process.9. Do not mix reagents with those from other lots. Storage and validity1.Storage: 2-8℃.2.validity: six months
标准
2015.07.22
Cell:抗体注射疗法可实现癌症的长期免疫保护在治疗肿瘤的过程中我们可能会使用被动注射肿瘤特异性抗体的方法。这种方法的原理是依靠抗体的中和效应将病原体进行识别,从而进一步被体内的巨噬细胞吞噬消灭,即"抗体依赖性的细胞毒性效应(ADCC)"。所以说,本质上这是一种短期的治疗方法。这一特点导致ADCC无法对肿瘤造成持续性的免疫效应。新的技术的出现帮我们寻找,鉴定肿瘤特异性的抗原,包括一些在肿瘤发生过程中新出现的抗原物质(neoantigens)。在最近的一项研究中,来自美国洛克菲勒大学的Jeffrey V. Ravetch与David J. DiLillo通过一种肿瘤特异性抗原的模型分析了被动注射抗体是否能够引起后续的免疫反应。
标准
2015.07.22
Cell: 脱发人士的福音,duang如果有一种可以治疗男性秃发的方法,但这种方法可能有一点疼。来自USC干细胞所,由Cheng-Ming Chuong领导的研究团队近日发现,在老鼠身上,通过以一种特殊的方式和密度拔200根头发,他们可以诱导出1,200根替代性的头发生长。这些研究结果发表在4月9号的Cell杂志上。此研究的研究方法的艰巨性和独特性深深的震撼了生物谷小编的心。"这是一个很好的例子,即 怎样把基础研究导向有潜在转化价值的工作,"USC Keck医学院病理系的主任Chuong说。"这项工作将发现潜在的新靶点治疗秃头症,脱发的一种。"这项研究开始于几年前,当时本文的第一作者,访问学者Chih-Chiang Chen从台湾国家Yang-Ming大学,退伍军人总医院来到了USC大学。作为一个皮肤科医生,Chen知道毛囊的损伤可以影响它周围的微环境,Chuong的实验室已经发现,这种微环境的改变反过来,也可以影响头发的再生。基于这些理论,他们认为,可能可以通过利用微环境来激活更多的毛囊。
标准
2015.07.22
人胰岛素样生长因子-1(IGF-1)酶联免疫分析试剂盒使用说明书本试剂盒仅供研究使用。检测范围: 96T8μg/L -240μg/L使用目的:本试剂盒用于测定人血清样本中胰岛素样生长因子-1(IGF-1)含量。实验原理本试剂盒应用双抗体夹心法测定标本中人胰岛素样生长因子-1(IGF-1)水平。用纯化的人胰岛素样生长因子-1(IGF-1)抗体包被微孔板,制成固相抗体,往包被单抗的微孔中依次加入胰岛素样生长因子-1(IGF-1),再与HRP 标记的胰岛素样生长因子-1(IGF-1)抗体结合,形成抗体-抗原-酶标抗体复合物,经过彻底洗涤后加底物TMB 显色。TMB 在HRP 酶的催化下转化成蓝色,并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的胰岛素样生长因子-1(IGF-1)呈正相关。用酶标仪在450nm 波长下测定吸光度(OD 值),通过标准曲线计算样品中人胰岛素样生长因子-1(IGF-1)浓度。试剂盒组成1 30 倍浓缩洗涤液20ml×1 瓶7 终止液6ml×1 瓶2 酶标试剂6ml×1 瓶8 标准品(480μg/L) 0.5ml×1 瓶3 酶标包被板12 孔×8 条9 标准品稀释液1.5ml×1 瓶4 样品稀释液6ml×1 瓶10 说明书1 份5 显色剂A 液6ml×1 瓶11 封板膜2 张6 显色剂B 液6ml×1/瓶12 密封袋1 个标本要求1. 血清:室温血液自然凝固10-20 分钟后,离心20 分钟左右(2000-3000 转/分)。仔细收集上清。保存过程中如有沉淀形成,应再次离心。2. 血浆:应根据标本的要求选择EDTA、柠檬酸钠或肝素作为抗凝剂,混合10-20 分钟后,离心20分钟左右(2000-3000 转/分)。仔细收集上清。保存过程中如有沉淀形成应再次离心。尿液:用无菌管收集。离心20 分钟左右(2000-3000 转/分)。仔细收集上清。保存过程中如有沉淀形成,应再次离心。胸腹水、脑脊液参照此实行。4.细胞培养上清:检测分泌性的成份时,用无菌管收集。离心20 分钟左右(2000-3000 转/分)。仔细收集上清。5.培养细胞:检测细胞内的成份时,用PBS(PH7.2-7.4)稀释细胞悬液,细胞浓度达到100 万/ml 左右。通过反复冻融或加入组织蛋白萃取试剂,以使细胞破坏并放出细胞内成份。离心20 分钟左右(2000-3000 转/分)。仔细收集上清。保存过程中如有沉淀形成,应再次离心。6.组织标本:切割标本后,称取重量。加入一定量的PBS,PH7.4。用液氮迅速冷冻保存备用。标本融化后仍然保持2-8℃的温度。加入一定量的PBS(PH7.4),或组织蛋白萃取试剂, 用手工或匀浆器将标本匀浆化。离心20 分钟左右(2000-3000 转/分)。仔细收集上清。分装后一份待检测,其余冷冻备用。7.标本采集后尽早进行提取,提取按相关文献进行,提取后应尽快进行实验。若不能马上进行试验,可将标本放于-20℃保存,但应避免反复冻融8.不能检测含NaN3 的样品,因NaN3 抑制辣根过氧化物酶的(HRP)活性。操作步骤1. 标准品的稀释:本试剂盒提供原倍标准品一支,用户可按照下列图表在小试管中进行稀释。240μg/L 5 号标准品150μl 的原倍标准品加入150μl 标准品稀释液120μg/L 4 号标准品150μl 的5 号标准品加入150μl 标准品稀释液60μg/L 3 号标准品150μl 的4 号标准品加入150μl 标准品稀释液30μg/L 2 号标准品150μl 的3 号标准品加入150μl 标准品稀释液15μg/L 1 号标准品150μl 的2 号标准品加入150μl 标准品稀释液2. 加样:分别设空白孔(空白对照孔不加样品及酶标试剂,其余各步操作相同)、标准孔、待测样品孔。在酶标包被板上标准品准确加样50μl,待测样品孔中先加样品稀释液40μl,然后再加待测样品10μl(样品最终稀释度为5 倍)。加样将样品加于酶标板孔底部,尽量不触及孔壁,轻轻晃动混匀。3. 温育:用封板膜封板后置37℃温育30 分钟。4. 配液:将30 倍浓缩洗涤液用蒸馏水30 倍稀释后备用5. 洗涤:小心揭掉封板膜,弃去液体,甩干,每孔加满洗涤液,静置30 秒后弃去,如此重复5 次,拍干。6. 加酶:每孔加入酶标试剂50μl,空白孔除外。7. 温育:操作同3。8. 洗涤:操作同5。9. 显色:每孔先加入显色剂A50μl,再加入显色剂B50μl,轻轻震荡混匀,37℃避光显色10 分钟.10. 终止:每孔加终止液50μl,终止反应(此时蓝色立转黄色)。11. 测定:以空白空调零,450nm 波长依序测量各孔的吸光度(OD 值)。测定应在加终止液后15 分钟以内进行。操作程序总结:计算以标准物的浓度为横坐标,OD 值为纵坐标,在坐标纸上绘出标准曲线,根据样品的OD 值由标准曲线查出相应的浓度;再乘以稀释倍数;或用标准物的浓度与OD 值计算出标准曲线的直线回归方程式,将样品的OD 值代入方程式,计算出样品浓度,再乘以稀释倍数,即为样品的实际浓度。注意事项1.试剂盒从冷藏环境中取出应在室温平衡15-30 分钟后方可使用,酶标包被板开封后如未用完,板条应装入密封袋中保存。2.浓洗涤液可能会有结晶析出,稀释时可在水浴中加温助溶,洗涤时不影响结果。3.各步加样均应使用加样器,并经常校对其准确性,以避免试验误差。一次加样时间最好控制在5 分钟内,如标本数量多,推荐使用排枪加样。4. 请每次测定的同时做标准曲线,最好做复孔。如标本中待测物质含量过高(样本OD 值大于标准品孔第一孔的OD 值),请先用样品稀释液稀释一定倍数(n 倍)后再测定,计算时请最后乘以总稀释倍数(×n×5)。5. 封板膜只限一次性使用,以避免交叉污染。6.底物请避光保存。7.严格按照说明书的操作进行,试验结果判定必须以酶标仪读数为准.8.所有样品,洗涤液和各种废弃物都应按传染物处理。9.本试剂不同批号组分不得混用。保存条件及有效期1.试剂盒保存:;2-8℃。2.有效期:6 个月
标准
2015.07.20
酒精代谢原来有特殊基因近日,发表于国际杂志PNAS上的一篇研究论文中,来自国外的研究人员通过对果蝇和人类机体进行研究揭开了Rsu1基因和其对酒精消耗影响程度之间的关系,该研究或可有效帮助个体抑制酒精滥用。很多年以来,大量实例研究都阐明了酗酒中存在的遗传组分,相比其它家庭的儿童而言,酗酒者显然更容易对自己的孩子构成风险;这项研究中研究人员就通过研究来寻找和酗酒相关的风险基因,首先研究人员研究了酒精对果蝇的影响,结果发现,昆虫受酒精影响的表现和人类一样,即少量的酒精都会增加机体的活性,减少协调性,而大量的酒精直接会让个体处于睡眠之中。同时研究果蝇也可以阐明成瘾症状的类型,研究者对果蝇基因组进行了分析发现,果蝇机体可以产生一种特殊蛋白来对Rsu1基因产生反应,这似乎就可以控制果蝇被酒精所影响的程度,而研究者发现通过修饰和基因相关连的蛋白的产生或可使得果蝇被酒精摄入的影响降低。下一步研究者希望通过对人类机体进行研究,因为人类机体中含有和果蝇一样的Rsu1基因,研究者招募了将近1400名非成瘾的未成年人进行研究,其利用大脑扫描结果来揭示研究对象大脑受酒精激发的程度,通过对比研究对象大脑的激发程度和饮酒史的关系,研究者发现个体的饮酒习惯和Rsu1基因影响酒精代谢调节的程度直接相关,即基因影响越小的个体饮酒量趋于越少,这就表明,抑制人类机体中Rsu1基因的表达或可帮助抑制个体的酒精依赖性,而研究者指出,Rsu1基因似乎对个体的终生饮酒习惯还有一定的影响。最后研究者表示,开发一种改变Rsu1基因表达的药物似乎并不太现实,研究者希望后期可以进行更多的研究来通过修饰Rsu1基因表达的蛋白来间接地抑制个体的酒精依赖性,这或许可以帮助有效开发个体酒精滥用的新型策略。
标准
2015.07.20
纳米微球高效高剂量狙杀癌细胞刊登在国际杂志Advanced Materials上的一项研究报告中,来自加尼弗尼亚大学的研究人员通过研究开发了一种新型的纳米粒子,其可以在一类特殊蛋白存在的情况下释放药物发挥作用,而特殊蛋白则可以驱动癌症发生转移;文章中研究者开发了一种药物运输系统,其可以有效抑制癌症的发展。研究者Cassandra Callmann说道,我们以一种小型分子为基础构建了纳米尺度的载体,其可以找到肿瘤并且释放所装载的药物;而这种药物运输系统可以利用一种名为基质金属蛋白酶(MMPs)的特殊酶类蛋白,这种特殊的酶类蛋白在多种癌症中都非常丰富,MMPs可以“撕裂”细胞膜,使得癌细胞可以逃脱而进入机体其他部位引发癌症转移。研究者开发了一种小型的装载抗癌药物紫杉酚的微型球状结构,并且覆盖以肽类外壳,MMPs可以撕开外壳释放其中所装载的药物,外壳碎片可以形成一种漏网,其就可以包裹药物使其对肿瘤进行稳定地作用。研究者表示,这项研究对于后期开发癌症诊断以及治疗的新型策略非常重要,为了将药物包裹如微型球体结构中,研究者进行了一定的化学处理,事实证明一系列原子对于药物分子的作用效力非常必要,而这也意味着癌症药物在达到肿瘤之前,在其流经的循环系统中是处于失活状态的,这也就避免了药物副作用的发生。这种癌症药物的包裹方式相比临床用癌症药物剂量而言可以在安全范围内增加至16倍,在预试验结果,研究者发现这种新型微型球状结构包裹的纳米药物可以在两周内有效抑制小鼠机体中肿瘤的生长。研究者表示,这项研究对于开发新型的药物运输系统来用作其它诊断和疾病治疗非常重要,而本文研究所提供的平台后期或将用于对其它多种类型癌症疗法的开发。后期研究者将继续对包裹药物的外壳进行修饰来为药物提供更好的保护手段,从而避免肝脏等其它器官对药物进行吸收,进而增加药物达到肿瘤的剂量以及药物的作用效果。
标准
2015.07.20
Human PARP-1 FOR RESEARCH USE ONLY Assay range:12μmol/L - 450μmol/L 96 determinationsPurposeThis kit allows for the determination of PARP-1 concentrations in Human serum, cell culture supernates and other biological fluids Principle of the assayThe kit assay Human PARP-1 level in the sample, use Purified Human PARP-1 antibody to coat microtiter plate wells, make solid-phase antibody, then add PARP-1 to wells, Combined PARP-1 antibody which With HRP labeled, become antibody - antigen - enzyme-antibody complex, after washing Completely, Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of Human PARP-1 in the samples is then determined by comparing the O.D. of the samples to the standard curve.Materials provided with the kit1 wash solution 20ml×1bottle 7 Stop Solution 6ml×1 bottle 2 HRP-Conjugate reagent 6ml×1 bottle 8 Standard(800μmol/L) 0.5ml×1 bottle 3 Microelisa stripplate 12well×8strips 9 Standard diluent 1.5ml×1bottle 4 Sample diluent 6ml×1 bottle 10 Instruction 1 5 Chromogen Solution A 6ml×1 bottle 11 Closure plate membrane 2 6 Chromogen Solution B 6ml×1 bottle 12 Sealed bags 1 Specimen requirements1. extract as soon as possible after Specimen collection,and according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can’t, specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles.2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.Assay procedure1. Dilute and add sample:Dilute Original density Standard as follow table:400μmol/L 5 Standard 150μl Original density Standard+150μl Standard diluent 200μmol/L 4 Standard 150μl 5 Standard+150μl Standard diluent 100μmol/L 3 Standard 150μl 4 Standard+150μl Standard diluent 50μmol/L 2 Standard 150μl 3 Standard +150μl Standard diluent 25μmol/L 1 Standard 150μl 2 Standard +150μl Standard diluent 2. Add sample: Set blank wells separately (blank comparison wells don’t add sample and HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution 40μl to testing sample well, then add testing sample 10μl (sample final dilution is 5-fold), add sample to wells , don’t touch the well wall as far as possible, and Gently mix.3. Incubate: After closing plate with Closure plate membrane , incubate for 30 min at 37℃.4. Configurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.5. Washing: Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.6. Add enzyme: Add HRP-Conjugate reagent 50μl to each well, except blank well. 7. Incubate: Operation with 3.8. Washing: Operation with 5.9. Color: Add Chromogen Solution A 50ul and Chromogen Solution B 50ul to each well, evade the light preservation for 10 min at 37℃10. Stop the reaction: Add Stop Solution50μl to each well, Stop the reaction(the blue color change to yellow color).11. Assay: take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min.Steps descriptionStandard, Sample diluent Add Standard, Sample diluent, incubate for 30 min at 37℃. Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37℃. Wash 5 times,Add Chromogen Solution A and B, incubate for 10 min at 37℃. Add Stop Solution Read absorbance at 450nm within 15 min calculate CalculateTake the standard density as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find out the corresponding density according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation of the standard curve with the standard density and the OD value ,with the sample OD value in the equation, calculate the sample density, multiplied by the dilution factor, the result is the sample actual density.Important notes1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.2. washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result.3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. add sample within 5 min, if the number of sample is much , recommend to use Volley .4. if the testing material content is excessively higher (The sample OD is bigger than the first standard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilution factor.(×n×5).5. Closure plate membrane only limits the disposable use, to avoid cross-contamination.6. The substrate evade the light preservation.7. Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.8. All samples, washing buffer and each kind of reject should according to infective material process.9. Do not mix reagents with those from other lots. Storage and validity1.Storage: 2-8℃.2.validity: six months
标准
2015.07.15
Human Nephrin FOR RESEARCH USE ONLY Assay range:0.5μg/L -12μg/L 96 determinationsPurposeThis kit allows for the determination of Nephrin concentrations in Human serum, cell culture supernates and other biological fluids Principle of the assayThe kit assay Human Nephrin level in the sample, use Purified Human Nephrin antibody to coat microtiter plate wells, make solid-phase antibody, then add Nephrin to wells, Combined Nephrin antibody which With HRP labeled, become antibody - antigen - enzyme-antibody complex, after washing Completely, Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of Human Nephrin in the samples is then determined by comparing the O.D. of the samples to the standard curve.Materials provided with the kit1 wash solution 20ml×1bottle 7 Stop Solution 6ml×1 bottle 2 HRP-Conjugate reagent 6ml×1 bottle 8 Standard(24μg/L) 0.5ml×1 bottle 3 Microelisa stripplate 12well×8strips 9 Standard diluent 1.5ml×1bottle 4 Sample diluent 6ml×1 bottle 10 Instruction 1 5 Chromogen Solution A 6ml×1 bottle 11 Closure plate membrane 2 6 Chromogen Solution B 6ml×1 bottle 12 Sealed bags 1 Specimen requirements1. extract as soon as possible after Specimen collection,and according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can’t, specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles.2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.Assay procedure1. Dilute and add sample:Dilute Original density Standard as follow table:12μg/L 5 Standard 150μl Original density Standard+150μl Standard diluent 6μg/L 4 Standard 150μl 5 Standard+150μl Standard diluent 3μg/L 3 Standard 150μl 4 Standard+150μl Standard diluent 1.5pg/ml 2 Standard 150μl 3 Standard +150μl Standard diluent 0.75pg/ml 1 Standard 150μl 2 Standard +150μl Standard diluent 2. Add sample: Set blank wells separately (blank comparison wells don’t add sample and HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution 40μl to testing sample well, then add testing sample 10μl (sample final dilution is 5-fold), add sample to wells , don’t touch the well wall as far as possible, and Gently mix.3. Incubate: After closing plate with Closure plate membrane , incubate for 30 min at 37℃.4. Configurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.5. Washing: Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.6. Add enzyme: Add HRP-Conjugate reagent 50μl to each well, except blank well. 7. Incubate: Operation with 3.8. Washing: Operation with 5.9. Color: Add Chromogen Solution A 50ul and Chromogen Solution B 50ul to each well, evade the light preservation for 10 min at 37℃10. Stop the reaction: Add Stop Solution50μl to each well, Stop the reaction(the blue color change to yellow color).11. Assay: take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min.Steps descriptionStandard, Sample diluent Add Standard, Sample diluent, incubate for 30 min at 37℃. Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37℃. Wash 5 times,Add Chromogen Solution A and B, incubate for 10 min at 37℃. Add Stop Solution Read absorbance at 450nm within 15 min calculate CalculateTake the standard density as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find out the corresponding density according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation of the standard curve with the standard density and the OD value ,with the sample OD value in the equation, calculate the sample density, multiplied by the dilution factor, the result is the sample actual density.Important notes1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.2. washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result.3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. add sample within 5 min, if the number of sample is much , recommend to use Volley .4. if the testing material content is excessively higher (The sample OD is bigger than the first standard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilution factor.(×n×5).5. Closure plate membrane only limits the disposable use, to avoid cross-contamination.6. The substrate evade the light preservation.7. Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.8. All samples, washing buffer and each kind of reject should according to infective material process.9. Do not mix reagents with those from other lots. Storage and validity1.Storage: 2-8℃.2.validity: six months
标准
2015.07.15
美生物医学研究使用动物数量创历史新低美国农业部(USDA)近日发布的最新统计数据显示,去年,用于该国生物医学研究的联邦监管动物数量降至1972年收集数据以来的最低谷。2014年,约83.4万只兔子、非人类灵长目动物和其他监管动物被用于研究,而上世纪70年代初期,这一数字约为150万。自1993年以来,这些动物的用量就开始呈下滑趋势,2013~2014年间下降了6%。2008年,USDA开始在其官网上张贴实验动物用量。到现在,总使用量下降了17%。不过,这一数字不包括大部分老鼠、鸟类和鱼类。这些动物占实验用动物的98%,但未被1966年的《动物福利法》(AWA)纳入在内。“这将是一个长期持续的趋势,并且没有迹象显示将减慢。事实上,它还在增速。”英国支持使用实验动物的团体“谈及研究”负责人Tom Holder说。而善待动物组织(PETA)实验室监管资深专家Alka Chandna表示,动物权益保护人士“非常高兴”。2013~2014年,每种AWA涉及的动物的研究使用量几乎都出现下滑。这段时间里,狗的用量下降了12%、兔子和豚鼠均下降了11%、非人类灵长目动物减少了10%。只有“所有其他物种类”的用量出现了增加,这类动物包括雪貂、松鼠和一些啮齿类动物。2013~2014年,这类动物用量增加了25%。自2008年以来,猫的用量增加了4%,但在2013~2014年则减少了13%。USDA发言人表示,该机构将不会推测何种因素导致了这一发展趋势,但Holder和Chandna指出了一些因素:计算机模型和组织培养的发展、动物实验外包、成本增加及后勤运输方面的挑战等。另外,公众对实验动物的态度发生了变化,调查显示,目前一半的美国人反对使用实验动物。公众态度也促使政府行动起来。英国于去年公布一项计划,拟通过替代等方式减少科研中对动物的使用,并敦促科研人员改善实验过程,减轻实验动物的痛苦。
标准
2015.07.15
人CD3分子(CD3)酶联免疫分析试剂盒使用说明书本试剂盒仅供研究使用。检测范围: 96T2.5 IU/ml -80 IU/ml使用目的:本试剂盒用于测定人血清、血浆及相关液体样本中CD3 分子(CD3)含量。实验原理本试剂盒应用双抗体夹心法测定标本中人CD3 分子(CD3)水平。用纯化的人CD3 分子(CD3)抗体包被微孔板,制成固相抗体,往包被单抗的微孔中依次加入CD3 分子(CD3),再与HRP 标记的CD3 分子(CD3)抗体结合,形成抗体-抗原-酶标抗体复合物,经过彻底洗涤后加底物TMB 显色。TMB 在HRP 酶的催化下转化成蓝色,并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的CD3 分子(CD3)呈正相关。用酶标仪在450nm 波长下测定吸光度(OD 值),通过标准曲线计算样品中人CD3 分子(CD3)浓度。试剂盒组成1 30 倍浓缩洗涤液20ml×1 瓶7 终止液6ml×1 瓶2 酶标试剂6ml×1 瓶8 标准品(160 IU/ml) 0.5ml×1 瓶3 酶标包被板12 孔×8 条9 标准品稀释液1.5ml×1 瓶4 样品稀释液6ml×1 瓶10 说明书1 份5 显色剂A 液6ml×1 瓶11 封板膜2 张6 显色剂B 液6ml×1/瓶12 密封袋1 个标本要求1. 血清:室温血液自然凝固10-20 分钟后,离心20 分钟左右(2000-3000 转/分)。仔细收集上清。保存过程中如有沉淀形成,应再次离心。2. 血浆:应根据标本的要求选择EDTA、柠檬酸钠或肝素作为抗凝剂,混合10-20 分钟后,离心20分钟左右(2000-3000 转/分)。仔细收集上清。保存过程中如有沉淀形成应再次离心。尿液:用无菌管收集。离心20 分钟左右(2000-3000 转/分)。仔细收集上清。保存过程中如有沉淀形成,应再次离心。胸腹水、脑脊液参照此实行。4.细胞培养上清:检测分泌性的成份时,用无菌管收集。离心20 分钟左右(2000-3000 转/分)。仔细收集上清。5.培养细胞:检测细胞内的成份时,用PBS(PH7.2-7.4)稀释细胞悬液,细胞浓度达到100 万/ml 左右。通过反复冻融或加入组织蛋白萃取试剂,以使细胞破坏并放出细胞内成份。离心20 分钟左右(2000-3000 转/分)。仔细收集上清。保存过程中如有沉淀形成,应再次离心。6.组织标本:切割标本后,称取重量。加入一定量的PBS,PH7.4。用液氮迅速冷冻保存备用。标本融化后仍然保持2-8℃的温度。加入一定量的PBS(PH7.4),或组织蛋白萃取试剂, 用手工或匀浆器将标本匀浆化。离心20 分钟左右(2000-3000 转/分)。仔细收集上清。分装后一份待检测,其余冷冻备用。7.标本采集后尽早进行提取,提取按相关文献进行,提取后应尽快进行实验。若不能马上进行试验,可将标本放于-20℃保存,但应避免反复冻融8.不能检测含NaN3 的样品,因NaN3 抑制辣根过氧化物酶的(HRP)活性。操作步骤1. 标准品的稀释:本试剂盒提供原倍标准品一支,用户可按照下列图表在小试管中进行稀释。80 IU/ml 5 号标准品150μl 的原倍标准品加入150μl 标准品稀释液40 IU/ml 4 号标准品150μl 的5 号标准品加入150μl 标准品稀释液20 IU/ml 3 号标准品150μl 的4 号标准品加入150μl 标准品稀释液10 IU/ml 2 号标准品150μl 的3 号标准品加入150μl 标准品稀释液5 IU/ml 1 号标准品150μl 的2 号标准品加入150μl 标准品稀释液2. 加样:分别设空白孔(空白对照孔不加样品及酶标试剂,其余各步操作相同)、标准孔、待测样品孔。在酶标包被板上标准品准确加样50μl,待测样品孔中先加样品稀释液40μl,然后再加待测样品10μl(样品最终稀释度为5 倍)。加样将样品加于酶标板孔底部,尽量不触及孔壁,轻轻晃动混匀。3. 温育:用封板膜封板后置37℃温育30 分钟。4. 配液:将30 倍浓缩洗涤液用蒸馏水30 倍稀释后备用5. 洗涤:小心揭掉封板膜,弃去液体,甩干,每孔加满洗涤液,静置30 秒后弃去,如此重复5 次,拍干。6. 加酶:每孔加入酶标试剂50μl,空白孔除外。7. 温育:操作同3。8. 洗涤:操作同5。9. 显色:每孔先加入显色剂A50μl,再加入显色剂B50μl,轻轻震荡混匀,37℃避光显色10 分钟.10. 终止:每孔加终止液50μl,终止反应(此时蓝色立转黄色)。11. 测定:以空白空调零,450nm 波长依序测量各孔的吸光度(OD 值)。测定应在加终止液后15 分钟以内进行。操作程序总结:计算以标准物的浓度为横坐标,OD 值为纵坐标,在坐标纸上绘出标准曲线,根据样品的OD 值由标准曲线查出相应的浓度;再乘以稀释倍数;或用标准物的浓度与OD 值计算出标准曲线的直线回归方程式,将样品的OD 值代入方程式,计算出样品浓度,再乘以稀释倍数,即为样品的实际浓度。注意事项1.试剂盒从冷藏环境中取出应在室温平衡15-30 分钟后方可使用,酶标包被板开封后如未用完,板条应装入密封袋中保存。2.浓洗涤液可能会有结晶析出,稀释时可在水浴中加温助溶,洗涤时不影响结果。3.各步加样均应使用加样器,并经常校对其准确性,以避免试验误差。一次加样时间最好控制在5 分钟内,如标本数量多,推荐使用排枪加样。4. 请每次测定的同时做标准曲线,最好做复孔。如标本中待测物质含量过高(样本OD 值大于标准品孔第一孔的OD 值),请先用样品稀释液稀释一定倍数(n 倍)后再测定,计算时请最后乘以总稀释倍数(×n×5)。5. 封板膜只限一次性使用,以避免交叉污染。6.底物请避光保存。7.严格按照说明书的操作进行,试验结果判定必须以酶标仪读数为准.8.所有样品,洗涤液和各种废弃物都应按传染物处理。9.本试剂不同批号组分不得混用。保存条件及有效期1.试剂盒保存:;2-8℃。2.有效期:6 个月
标准
2015.07.13
Human Bcma FOR RESEARCH USE ONLY Assay range:3.8pg/mL -180pg/mL 96 determinationsPurposeThis kit allows for the determination of BCMA concentrations in Human serum, cell culture supernates and other biological fluids Principle of the assayThe kit assay Human BCMA level in the sample, use Purified Human BCMA antibody to coat microtiter plate wells, make solid-phase antibody, then add BCMA to wells, Combined BCMA antibody which With HRP labeled, become antibody - antigen - enzyme-antibody complex, after washing Completely, Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of Human BCMA in the samples is then determined by comparing the O.D. of the samples to the standard curve.Materials provided with the kit1 wash solution 20ml×1bottle 7 Stop Solution 6ml×1 bottle 2 HRP-Conjugate reagent 6ml×1 bottle 8 Standard(320pg/mL) 0.5ml×1 bottle 3 Microelisa stripplate 12well×8strips 9 Standard diluent 1.5ml×1bottle 4 Sample diluent 6ml×1 bottle 10 Instruction 1 5 Chromogen Solution A 6ml×1 bottle 11 Closure plate membrane 2 6 Chromogen Solution B 6ml×1 bottle 12 Sealed bags 1 Specimen requirements1. extract as soon as possible after Specimen collection,and according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can’t, specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles.2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.Assay procedure1. Dilute and add sample:Dilute Original density Standard as follow table:160pg/mL 5 Standard 150μl Original density Standard+150μl Standard diluent 80pg/mL 4 Standard 150μl 5 Standard+150μl Standard diluent 40pg/mL 3 Standard 150μl 4 Standard+150μl Standard diluent 20pg/mL 2 Standard 150μl 3 Standard +150μl Standard diluent 10pg/mL 1 Standard 150μl 2 Standard +150μl Standard diluent 2. Add sample: Set blank wells separately (blank comparison wells don’t add sample and HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution 40μl to testing sample well, then add testing sample 10μl (sample final dilution is 5-fold), add sample to wells , don’t touch the well wall as far as possible, and Gently mix.3. Incubate: After closing plate with Closure plate membrane , incubate for 30 min at 37℃.4. Configurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.5. Washing: Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.6. Add enzyme: Add HRP-Conjugate reagent 50μl to each well, except blank well. 7. Incubate: Operation with 3.8. Washing: Operation with 5.9. Color: Add Chromogen Solution A 50ul and Chromogen Solution B 50ul to each well, evade the light preservation for 10 min at 37℃10. Stop the reaction: Add Stop Solution50μl to each well, Stop the reaction(the blue color change to yellow color).11. Assay: take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min.Steps descriptionStandard, Sample diluent Add Standard, Sample diluent, incubate for 30 min at 37℃. Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37℃. Wash 5 times,Add Chromogen Solution A and B, incubate for 10 min at 37℃. Add Stop Solution Read absorbance at 450nm within 15 min calculate CalculateTake the standard density as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find out the corresponding density according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation of the standard curve with the standard density and the OD value ,with the sample OD value in the equation, calculate the sample density, multiplied by the dilution factor, the result is the sample actual density.Important notes1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.2. washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result.3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. add sample within 5 min, if the number of sample is much , recommend to use Volley .4. if the testing material content is excessively higher (The sample OD is bigger than the first standard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilution factor.(×n×5).5. Closure plate membrane only limits the disposable use, to avoid cross-contamination.6. The substrate evade the light preservation.7. Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.8. All samples, washing buffer and each kind of reject should according to infective material process.9. Do not mix reagents with those from other lots. Storage and validity1.Storage: 2-8℃.2.validity: six months
标准
2015.07.13
血检可预测数年后糖尿病发展近日,MRC临床科学中心的科学家们首次表明1型糖尿病早期阶段的患者血液中有小分子循环,一个简单的血液测试就能检测出数年甚至几十年后的糖尿病生物标志物。“如果我们能早期识别和治疗病人那么就可以帮助他们避免继发性并发症的出现,从而可延长病人的寿命。”Mathieu Latreille说。目前医生诊断1型糖尿病主要依赖患者出现的临床症状,如口渴、体重减轻和视力模糊等症状。但是当免疫系统出现问题时病人身体就已经出现了损害,免疫系统会错误地将胰腺内细胞视为病原体。它攻击并破坏细胞,导致胰腺失去产生胰岛素的能力,而胰岛素控制着血液中糖的含量。病人可以通过注射胰岛素来管理他们的血糖水平,但即便这样仍有可能患并发症的风险,如失明、肾衰竭和心脏病。最近的研究表明,一种称为小分子核糖核酸的信使分子可以帮助医生诊断早期阶段的糖尿病。小分子核糖核酸带有跨细胞的遗传信息片段,并定期释放到血液中。在糖尿病患者体内,这些小分子核糖核酸的水平的循环会发生变化,这用一个简单的血液测试就可以检测到。但一个血液测试如果有可信性,它必须在特定在糖尿病中检测到这一变化。Latreille说,“小分子核糖核酸可以释放到任何组织中——眼睛、肾脏或腿中,但这些与胰腺健康没有任何关联。”到目前为止,科学家们一直无法确定小分子核糖核酸与胰腺的关联。但Latreille 的研究团队表明一种被称为小分子核糖核酸375的特定分子通过生产胰岛素的胰腺细胞释放。这些细胞通常含有大量的小分子核糖核酸375,它可以帮助控制细胞生长。在1型糖尿病患者体内,一旦细胞开始死亡小分子核糖核酸375就会大量释放到血液中。Latreille说,这可以成为医生诊断和治疗早期糖尿病的有用标志。下一步研究将是研究人员使用标志物可靠地预测出数年后谁会患糖尿病,然后制定出在什么阶段做什么样合适的治疗。
标准
2015.07.13
产品编号XY-0813R-HRP英文名称Anti-Casein/HRP中文名称辣根过氧化物酶标记的酪蛋白抗体别名Alpha casein; alpha-S1-casein; CASA; CASA1_Cow; Casein alpha s1; casein, alpha;casein, alpha-S1, included; Casoxin-D; CSN1; CSN1S1; Csna; MGC149368.说明书100ul研究领域细胞生物抗体来源Rabbit克隆类型Polyclonal交叉反应Cow,产品应用WB=1:50-200 ELISA=1:100-1000 IHC-P=1:50-200 IHC-F=1:50-200not yet tested in other applications.optimal dilutions/concentrations should be determined by the end user.分子量23kDa性状Lyophilized or Liquid浓度2mg/1ml免疫原Casein from bovine milk亚型IgG纯化方法affinity purified by Protein A储存Constituents: 0.01M PBS, pH 7.4 with 10 mg/mL BSA and 0.1% Gentamicin, 50% glycerol.液Or Lyophilized. Buffer = 0.01M PBS, pH 7.4 with 10 mg/mL BSA and 0.1% Gentamicin.Reconstitute with sterile distilled water.保存条件Store at -20 °C for one year. Avoid repeated freeze/thaw cycles. The lyophilizedantibody is stable at room temperature for at least one month and for greater thana year when kept at -20°C. When reconstituted in sterile pH 7.4 0.01M PBS or diluentof antibody the antibody is stable for at least two weeks at 2-4 °C.产品介绍background:Casein is the name for a family of related phosphoproteins (αS1, αS2, β, κ). Theseproteins are commonly found in mammalian milk, making up 80% of the proteins in cowmilk and between 20% and 45% of the proteins in human milk. Casein has a wide varietyof uses, from being a major component of cheese, to use as a food additive, to a binderfor safety matches. As a food source, casein supplies amino acids; carbohydrates;and two inorganic elements, calcium and phosphorus.Subunit:Casein contains a fairly high number of proline residues, which do not interact. Thereare also no disulfide bridges. As a result, it has relatively little tertiarystructure. It is relatively hydrophobic, making it poorly soluble in water. It isfound in milk as a suspension of particles called "casein micelles" which show onlylimited resemblance with surfactant-type micellae in a sense that the hydrophilicparts reside at the surface and they are spherical. However, in sharp contrast tosurfactant micelles, the interior of a casein micelle is highly hydrated. The caseinsin the micelles are held together by calcium ions and hydrophobic interactions.Several models account for the special conformation of casein in the micelles. Oneof them proposes the micellar nucleus is formed by several submicelles, the peripheryconsisting of microvellosities of κ-casein. Another model suggests the nucleus isformed by casein-interlinked fibrils. Finally, the most recent model proposes a doublelink among the caseins for gelling to take place. All three models consider micellesas colloidal particles formed by casein aggregates wrapped up in soluble κ-caseinmolecules.The isoelectric point of casein is 4.6. Since milk's pH is 6.6, casein has a negativecharge in milk. The purified protein is water insoluble. While it is also insolublein neutral salt solutions, it is readily dispersible in dilute alkalis and in saltsolutions such as sodium oxalate and sodium acetate.The enzyme trypsin can hydrolyze off a phosphate-containing peptone. It is used toform a type of organic adhesive.Important Note:This product as supplied is intended for research use only, not for use in human,therapeutic or diagnostic applications.酪蛋白(casein)是牛乳包括牛初乳的主要成分,通常在分离提取乳清过程中可得到大量的酪蛋白,它具有各种必需氨基酸,且组分合理,是一种优质的动物蛋白。然而,由于人体尤其是儿童对牛乳酪蛋白的分解能力较弱,牛乳酪蛋白的应用并不广泛。近来国外文献报道,牛乳酪蛋白短链肽除了易吸收外,还具有某些重要的生物学功能,提示着酪蛋白除单纯营养作用之外的广阔应用前景。
标准
2015.07.10
Human β-D-glucan (BG) ELISA KitFOR RESEARCH USE ONLY. Not for clinical diagnosis useIntroductionl For the quantitative determination of Human BG concentrations. l Detection of species: Humanl Detection medium: serum, plasma, tissue homogenates, cell culture supernates Principle of testThe kit is for the quantitative level of BG in the sample, adopt purified Human BG to coat microtiter plate, make solid-phase antibody, then add BG to wells, Combine BG antibody with labeled HRP to form antibody-antigen -enzyme-antibody complex, after washing completely, add TMB substrate solution, TMB substrate becomes blue color at HRP enzyme-catalyzed, reaction is terminated by the addition of a stop solution and the color change is measured at a wavelength of 450 nm. The concentration of BG in the samples is then determined by comparing the O.D. of the samples to the standard curve. Composition of the kitReagent Quantity Microelisa Stripplate 12well×8strips Standard: 32μg/mL 1×0.5ml Standard Diluent 1×1.5ml HRP-Conjugate Reagent 1×6ml Sample Diluent 1×6ml Chromogen Solution A 1×6ml Chromogen Solution B 1×6ml Stop Solution 1×6ml Wash Solution 1×20ml×30 fold User manual 1 Adhesive Strip 2 Storage conditionsl The unopened kit shall be stored at [2-8 ℃] .l For opened kit can be stored at [2-8 ℃] for up to 1 month. If not be used recently, the standard should be kept in -20 ℃. Washing methodl Manually washing method: shake away the remained liquid in the enzyme plates; place some bibulous papers on the test-bed, and flap the plates on the upside down strongly. Inject at least 0.35ml after-dilution washing solution into the well, and marinate 1~2 minutes. Repeat this process according to your requirements. l Automatic washing method: if there is automatic washing machine, it should only be used in the test when you are quite familiar with its function and performance. Specimen requirements1. Serum-coagulation at room temperature for 10-20 min,centrifuge at the speed of 2000-3000 rpm for 20-min. Remove supernatant, if precipitation appeared, Centrifuge again.2. Plasma-use suited EDTA or citrate plasma as an anticoagulant, centrifuge at the speed of 2000-3000 rpm for 20-min. Remove supernatant, if precipitation appeared, centrifuge again.3. Urine-collect sue a sterile container, centrifuge at the speed of 2000-3000 rpm for 20-min. Remove supernatant, if precipitation appeared, Centrifuge again. The Operation of Hydrothorax and cerebrospinal fluid reference to it.4. Cell culture supernatant-detect secretory components, Remove particulates by centrifugation for 20-min at the speed of 2000-3000 rpm. Remove supernatant detect the composition of cells, dilute cell suspension with PBS (PH7.2-7.4), Cell concentration reached 1 million / ml, repeated freeze-thaw cycles, damage cells and release of intracellular components, centrifugation 20-min at the speed of 2000-3000 rpm. remove supernatant, If precipitation appeared, Centrifugal again.5. Tissue samples- After cutting samples, check the weight, Pipette PBS(PH7.2-7.4), Rapidly frozen with liquid nitrogen, maintain samples at 2-8℃ after melting, Pipette PBS(PH7.4), homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 rpm. Remove supernatant.6. Extract as soon as possible after Samples collection, and should be tested as soon as possible after the extraction. If not, samples can be kept in -20 ℃ to preserve. Avoid repeated freeze-thaw cycles.7. Can' t detect the sample which contain NaN3, because NaN3 inhibits HRP active. Assay procedure Step 1: Standard: Bring all reagents to room temperature.Dilute the standard:Pipette 50μl standard dilution in each tube. Pipette 100μl standard (32μg/mL) in the first tube. And take out 100μl from the first tube into the second. Pipette 50μl from the second tube to the third tube and produce dilution series as below.Repeat each of the concentration to get the mean value of each well. Tube 0 1 2 3 4 5 μg/mL 32 16 8 4 2 1 Step 2: Prepare sample: Set blank wells separately (blank comparison wells don't add sample and HRP-Conjugate reagent, other each step operation is same). Pipette Sample dilution 40μl to testing sample well, then add testing sample 10μl (sample final dilution is 5-fold), Pipette sample to wells, don’t touch the well wall as far as possible, and mix gently.Step 3: Incubate: Cover with the adhesive strip provided, incubate for 30 min at 37℃.Step 4: Configurate liquid: Dilute wash solution 30-fold (or 20-fold) with distilled water.Step 5: Washing: Uncover the adhesive strip, discard liquid, Pipette washing buffer to every well, still for 30s then drain, repeat 5 times.Step 6: Add enzyme: Pipette HRP-Conjugate reagent 50μl to each well, except blank well. Step 7: Incubate: Operation with 3.Step 8: Washing: Operation with 5.Step 9: Color: Pipette Chromogen Solution A 50ul and Chromogen Solution B to each well, avoid the light preservation for 15 min at 37℃Step 10: Stop the reaction: Pipette Stop Solution 50μl to each well, Stop the reaction (the blue change to yellow).Step 11: Assay: take blank well as zero, Read absorbance at 450nm after pipetteing Stop Solution within 15min. Judgment of assay resultTake the standard concentration as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find out the corresponding concentration according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation of the standard curve with the standard concentration and the OD value, with the sample OD value in the equation, calculate the sample concentration, multiplied by the dilution factor, the result is the sample actual concentration. Graphical Representation as following: DESCRIPTION1. The standard curve is drawn under ideal conditions, and is just for reference rather than the actual standard curve diagram of the kit.2. It is advisable to establish proper assay data and standard curve according to respective laboratory conditions. DETECTION RANGEDetection range of the kit is: 0.4μg/mL -20μg/mL Expiration Twelve months [see label on the outer box for the specific date]. Attentionl The kit takes out from the refrigeration should be balanced 15-30 minutes in the room temperature, if the coated ELISA plates have not been used up after opening, the plate should be stored in sealed bag.l Washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilution. Washing does not affect the result.l Pipette sample with pipettors each step, and proofread its accuracy frequently, avoids the experimental error. Pipette sample within 5 min, if the number of sample is much, recommend using multichannel pipettor.l If the testing material content is excessively high (The sample OD is higher than the first standard well),please dilute sample (n-fold).l Adhesive Strip only limits the disposable use to avoid cross-contamination.l The substrate should be preserved evade the light.l Please refer to use instruction strictly. The test result determination must take the microtiter plate reader as a standard.l All samples, washing buffer and each kind of reject should refer to infective material process.l Do not mix reagents with those from other lots.
标准
2015.07.10
基因治疗使小鼠恢复听力,遗传性耳聋有望治愈波士顿儿童医院和哈佛医学院的研究人员通过基因治疗为“耳聋”小鼠模型恢复了听力,其研究成果于7月8日在线发表在Science Translational Medicine杂志上,这将推动基因治疗应用于遗传性耳聋治疗的进程。波士顿儿童医院的科学家Jeffrey Holt说:“我们的基因治疗方法尚未用于临床实验,还需再优化,并在不久的将来将其用于治疗耳聋患者。”目前已发现超过70个基因与耳聋相关(实际上,华大基因目前可以对非综合征型与综合征型耳聋常见与罕见的127 个致病基因进行检测)。研究人员重点研究了TMC1基因,因为它是遗传性耳聋中最为常见的致病基因,约占耳聋患者人群的4-8%。该基因编码的蛋白在听力过程中起关键作用,帮助将声音转化成电信号然后传导到大脑。研究人员在两种突变型小鼠模型中测试基因治疗的效果。在其中的一种突变型小鼠中,TMC1基因完全被敲除。这种小鼠模型对于研究携带TMC1基因隐性致病突变的人群很有帮助,如果孩子分别从父母那里遗传了TMC1基因的致病突变,通常会在2岁左右完全丧失听力。另一种突变型小鼠叫Beethoven,是一种很特别的TMC1突变体,这种突变型小鼠会因为基因突变导致一个氨基酸发生变化。这种小鼠模型对于研究因TMC1基因显性遗传而致病的耳聋患者很有帮助。显性遗传性耳聋比隐性遗传性耳聋相对少见,这种类型的耳聋患者只需一个拷贝的突变就会使孩子逐渐丧失听力,一般会在10-15岁发病。为了将正常的基因导入小鼠体内,研究团队将正常的基因与启动子序列一起插入到一种名为1号腺伴随病毒或AAV1的工程病毒载体中,只有在内耳的感觉细胞(又称为毛细胞)中,启动子才会启动基因的表达。包含正常TMC1基因的AAV1病毒载体被注入内耳之后,可以发现:1.在隐性遗传性耳聋小鼠模型中,基因治疗使小鼠的毛细胞恢复听觉功能,能够对声音做出反应,并且可以监测到其产生的电流,脑干的听觉相关区域也恢复了功能。2.最重要的是,耳聋小鼠恢复了听力。为了测试其听力,研究人员将小鼠置于一个惊吓测试盒中,然后突然发出响亮的声音,TMC1突变型小鼠对声音没有反应,仅仅是坐着,但经过基因治疗后,在听到声音后它们会像正常小鼠一样跳起来。3.对于显性遗传性耳聋小鼠模型,使用相关的基因TMC2进行基因治疗后,在细胞和大脑组织水平上看是成功的,在惊吓测试中小鼠的听力功能有一定程度的恢复。
标准
2015.07.10
Rat D-Dimer (D2D)FOR RESEARCH USE ONLYAssay range:0.1ng/ml – 4.0ng/ml 96 determinationsPurposeThis kit allows for the determination of D2D concentrations in Rat serum, cell culturesupernates and other biological fluidsPrinciple of the assayThe kit assay Rat D2D level in the sample, use Purified Rat D2D antibody to coatmicrotiter plate wells, make solid-phase antibody, then add D2D to wells, Combined D2Dantibody which With HRP labeled, become antibody - antigen - enzyme-antibody complex, afterwashing Completely, Add TMB substrate solution,TMB substrate becomes blue color At HRPenzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and thecolor change is measured spectrophotometrically at a wavelength of 450 nm. Theconcentration of Rat D2D in the samples is then determined by comparing the O.D. of thesamples to the standard curve.Materials provided with the kit1 wash solution 20ml×1bottle 7 Stop Solution 6ml×1 bottle2 HRP-Conjugate reagent 6ml×1 bottle 8Standard(8.0ng/ml) 0.5ml×1 bottle3 Microelisa stripplate 12well×8strips 9 Standard diluent 1.5ml×1bottle4 Sample diluent 6ml×1 bottle 10 Instruction 15 Chromogen Solution A 6ml×1 bottle 11 Closure plate 2IBL International GmbHFlughafenstra?e 52a 22335 Hamburg DEPhone: +49 (0)40-53 28 91-0 Fax: +49 (0)40-53 28 91-11IBL@IBL-International.com2membrane6 Chromogen Solution B 6ml×1 bottle 12 Sealed bags 1Specimen requirements1. extract as soon as possible after Specimen collection,and according to the relevantliterature, and should be experiment as soon as possible after the extraction. If it can’t,specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles.2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.Assay procedure1. Dilute and add sample:Dilute Original density Standard as follow table:4.0ng/ml 5 Standard 150μl Original density Standard+150μl Standard diluent2.0ng/ml 4 Standard 150μl 5 Standard+150μl Standard diluent1.0ng/ml 3 Standard 150μl 4 Standard+150μl Standard diluent0.5ng/ml 2 Standard 150μl 3 Standard +150μl Standard diluent0.25ng/ml 1 Standard 150μl 2 Standard +150μl Standard diluent2. Add sample: Set blank wells separately (blank comparison wells don’t add sample andHRP-Conjugate reagent, other each step operation is same). testing sample well. add Sampledilution 40μl to testing sample well, then add testing sample 10μl (sample final dilution is5-fold), add sample to wells , don’t touch the well wall as far as possible, and Gently mix.3. Incubate: After closing plate with Closure plate membrane , incubate for 30 min at 37℃.4. Configurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilledwater and reserve.5. Washing: Uncover Closure plate membrane, discard Liquid, dry by swing, add washingbuffer to every well, still for 30s then drain, repeat 5 times, dry by pat.6. Add enzyme: Add HRP-Conjugate reagent 50μl to each well, except blank well.7. Incubate: Operation with 3.8. Washing: Operation with 5.IBL International GmbHFlughafenstra?e 52a 22335 Hamburg DEPhone: +49 (0)40-53 28 91-0 Fax: +49 (0)40-53 28 91-11IBL@IBL-International.com39. Color: Add Chromogen Solution A 50ul and Chromogen Solution B 50ul to each well, evadethe light preservation for 10 min at 37℃10. Stop the reaction: Add Stop Solution50μl to each well, Stop the reaction(the blue colorchange to yellow color).11. Assay: take blank well as zero , Read absorbance at 450nm after Adding Stop Solution andwithin 15min.Steps descriptionStandard, Sample diluentAdd Standard, Sample diluent, incubate for 30 min at 37℃.Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37℃.Wash 5 times,Add Chromogen Solution A and B, incubate for 10 min at 37℃.Add Stop SolutionIBL International GmbHFlughafenstra?e 52a 22335 Hamburg DEPhone: +49 (0)40-53 28 91-0 Fax: +49 (0)40-53 28 91-11IBL@IBL-International.com4Read absorbance at 450nm within 15 mincalculateCalculateTake the standard density as the horizontal, the OD value for the vertical ,draw thestandard curve on graph paper, Find out the corresponding density according to the sampleOD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight lineregression equation of the standard curve with the standard density and the OD value ,with thesample OD value in the equation, calculate the sample density, multiplied by the dilution factor,the result is the sample actual density.Important notes1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes inthe room temperature, ELISA plates coated if has not use up after opened, the plate shouldbe stored in Sealed bag.2. washing buffer will Crystallization separation, it can be heated the water helps dissolvewhen dilute . Washing does not affect the result.IBL International GmbHFlughafenstra?e 52a 22335 Hamburg DEPhone: +49 (0)40-53 28 91-0 Fax: +49 (0)40-53 28 91-11IBL@IBL-International.com53. add Sample with sampler Each step, And proofread its accuracy frequently, avoids theexperimental error. add sample within 5 min, if the number of sample is much , recommendto use Volley .4. if the testing material content is excessively higher (The sample OD is bigger than the firststandard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilutionfactor.(×n×5).5. Closure plate membrane only limits the disposable use, to avoid cross-contamination.6. The substrate evade the light preservation.7. Please according to use instruction strictly, The test result determination must take themicrotiter plate reader as a standard.8. All samples, washing buffer and each kind of reject should according to infective materialprocess.9. Do not mix reagents with those from other lots.Storage and validity1.Storage: 2-8℃.2.validity: six months
标准
2015.07.08
Rat D-Dimer (D2D) ELISA KitFOR RESEARCH USE ONLYAssay range:0.1ng/ml – 4.0ng/ml 96 determinationsPurposeThis kit allows for the determination of D2D concentrations in Rat serum, cell culturesupernates and other biological fluidsPrinciple of the assayThe kit assay Rat D2D level in the sample, use Purified Rat D2D antibody to coatmicrotiter plate wells, make solid-phase antibody, then add D2D to wells, Combined D2Dantibody which With HRP labeled, become antibody - antigen - enzyme-antibody complex, afterwashing Completely, Add TMB substrate solution,TMB substrate becomes blue color At HRPenzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and thecolor change is measured spectrophotometrically at a wavelength of 450 nm. Theconcentration of Rat D2D in the samples is then determined by comparing the O.D. of thesamples to the standard curve.Materials provided with the kit1 wash solution 20ml×1bottle 7 Stop Solution 6ml×1 bottle2 HRP-Conjugate reagent 6ml×1 bottle 8Standard(8.0ng/ml) 0.5ml×1 bottle3 Microelisa stripplate 12well×8strips 9 Standard diluent 1.5ml×1bottle4 Sample diluent 6ml×1 bottle 10 Instruction 15 Chromogen Solution A 6ml×1 bottle 11 Closure platemembrane 2IBL International GmbHFlughafenstra?e 52a 22335 Hamburg DEPhone: +49 (0)40-53 28 91-0 Fax: +49 (0)40-53 28 91-11IBL@IBL-International.com26 Chromogen Solution B 6ml×1 bottle 12 Sealed bags 1Specimen requirements1. extract as soon as possible after Specimen collection,and according to the relevantliterature, and should be experiment as soon as possible after the extraction. If it can’t,specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles.2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.Assay procedure1. Dilute and add sample:Dilute Original density Standard as follow table:4.0ng/ml 5 Standard 150μl Original density Standard+150μl Standard diluent2.0ng/ml 4 Standard 150μl 5 Standard+150μl Standard diluent1.0ng/ml 3 Standard 150μl 4 Standard+150μl Standard diluent0.5ng/ml 2 Standard 150μl 3 Standard +150μl Standard diluent0.25ng/ml 1 Standard 150μl 2 Standard +150μl Standard diluent2. Add sample: Set blank wells separately (blank comparison wells don’t add sample andHRP-Conjugate reagent, other each step operation is same). testing sample well. add Sampledilution 40μl to testing sample well, then add testing sample 10μl (sample final dilution is5-fold), add sample to wells , don’t touch the well wall as far as possible, and Gently mix.3. Incubate: After closing plate with Closure plate membrane , incubate for 30 min at 37℃.4. Configurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilledwater and reserve.5. Washing: Uncover Closure plate membrane, discard Liquid, dry by swing, add washingbuffer to every well, still for 30s then drain, repeat 5 times, dry by pat.6. Add enzyme: Add HRP-Conjugate reagent 50μl to each well, except blank well.7. Incubate: Operation with 3.8. Washing: Operation with 5.IBL International GmbHFlughafenstra?e 52a 22335 Hamburg DEPhone: +49 (0)40-53 28 91-0 Fax: +49 (0)40-53 28 91-11IBL@IBL-International.com39. Color: Add Chromogen Solution A 50ul and Chromogen Solution B 50ul to each well, evadethe light preservation for 10 min at 37℃10. Stop the reaction: Add Stop Solution50μl to each well, Stop the reaction(the blue colorchange to yellow color).11. Assay: take blank well as zero , Read absorbance at 450nm after Adding Stop Solution andwithin 15min.Steps descriptionStandard, Sample diluentAdd Standard, Sample diluent, incubate for 30 min at 37℃.Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37℃.Wash 5 times,Add Chromogen Solution A and B, incubate for 10 min at 37℃.Add Stop SolutionIBL International GmbHFlughafenstra?e 52a 22335 Hamburg DEPhone: +49 (0)40-53 28 91-0 Fax: +49 (0)40-53 28 91-11IBL@IBL-International.com4Read absorbance at 450nm within 15 mincalculateCalculateTake the standard density as the horizontal, the OD value for the vertical ,draw thestandard curve on graph paper, Find out the corresponding density according to the sampleOD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight lineregression equation of the standard curve with the standard density and the OD value ,with thesample OD value in the equation, calculate the sample density, multiplied by the dilution factor,the result is the sample actual density.Important notes1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes inthe room temperature, ELISA plates coated if has not use up after opened, the plate shouldbe stored in Sealed bag.2. washing buffer will Crystallization separation, it can be heated the water helps dissolvewhen dilute . Washing does not affect the result.IBL International GmbHFlughafenstra?e 52a 22335 Hamburg DEPhone: +49 (0)40-53 28 91-0 Fax: +49 (0)40-53 28 91-11IBL@IBL-International.com53. add Sample with sampler Each step, And proofread its accuracy frequently, avoids theexperimental error. add sample within 5 min, if the number of sample is much , recommendto use Volley .4. if the testing material content is excessively higher (The sample OD is bigger than the firststandard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilutionfactor.(×n×5).5. Closure plate membrane only limits the disposable use, to avoid cross-contamination.6. The substrate evade the light preservation.7. Please according to use instruction strictly, The test result determination must take themicrotiter plate reader as a standard.8. All samples, washing buffer and each kind of reject should according to infective materialprocess.9. Do not mix reagents with those from other lots.Storage and validity1.Storage: 2-8℃.2.validity: six months
标准
2015.07.08
关键突变导致癌细胞代谢“重连”促进药物抵抗最近,来自艾默里大学的科学家发现在许多黑色素瘤中存在一个重要基因突变能够使癌细胞的代谢重新连线,使癌细胞的生长依赖于一种参与酮体生成的催化酶,这一发现为解决黑色素瘤细胞对靶向药物的抵抗,开发新的替代药物提供了深入见解,同时也部分解释了为什么这一突变在黑色素瘤细胞中频发。近日,相关研究结果发表在国际学术期刊molecular cell。 B-raf基因发生V600E突变在黑色素瘤细胞中非常常见,这一突变能够促进癌细胞生长,除了在黑色素瘤中存在,在一些结肠癌和甲状腺癌病例中也发现存在B-raf V600E突变。目前已经开发出一些针对B-raf V600E基因突变的靶向药物,但在临床实验中发现,在癌症得到明显改善之后,携带V600E基因突变的癌细胞都不可避免地产生药物抗性。 在这项研究中,研究人员发现B-raf V600E基因突变能够刺激癌细胞产生更多的HMG-CoA裂解酶,携带V600E突变的黑色素瘤细胞生长非常依赖于该酶,而其他的黑色素瘤细胞则不会。HMG-CoA裂解酶是酮体生成途径中一个重要酶,能够帮助机体在血糖水平较低时降解脂肪酸以获得能量。酮体生成途径能够受到低糖,高脂饮食刺激激活,通常发生于肝脏,但B-raf V600E基因突变启动了癌细胞中的酮体生成,以维持癌细胞生长存活。除此之外,研究人员还发现酮体生成途径的重要产物乙酰乙酸能够刺激携带B-raf V600E基因突变的癌细胞继续增殖。 总得来说,这项研究证明B-raf V600E基因突变能够使黑色素瘤细胞中的代谢途径重新连线,增强癌细胞对酮体生成途径的依赖性,这一发现对于解决黑色素瘤细胞对靶向药物的抵抗,开发新的替代药物具有重要意义。
标准
2015.07.08
大鼠超氧化物歧化酶(SOD)酶联免疫分析试剂盒使用说明书本试剂盒仅供研究使用。检测范围: 48T2.5 U/mL - 80 U/mL 使用目的:本试剂盒用于测定大鼠血清、血浆及相关液体样本中超氧化物歧化酶(SOD)含量。实验原理本试剂盒应用双抗体夹心法测定标本中大鼠超氧化物歧化酶(SOD)水平。用纯化的大鼠超氧化物歧化酶(SOD)抗体包被微孔板,制成固相抗体,往包被单抗的微孔中依次加入人超氧化物歧化酶(SOD),再与HRP标记的超氧化物歧化酶(SOD)抗体结合,形成抗体-抗原-酶标抗体复合物,经过彻底洗涤后加底物TMB显色。TMB在HRP酶的催化下转化成蓝色,并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的超氧化物歧化酶(SOD)呈正相关。用酶标仪在450nm波长下测定吸光度(OD值),通过标准曲线计算样品中大鼠超氧化物歧化酶(SOD)浓度。 试剂盒组成 1 20倍浓缩洗涤液 20ml×1瓶 7 终止液 3ml×1瓶 2 酶标试剂 3ml×1瓶 8 标准品(160 U/mL) 0.5ml×1瓶 3 酶标包被板 12孔×4条 9 标准品稀释液 1.5ml×1瓶 4 样品稀释液 3ml×1瓶 10 说明书 1份 5 显色剂A液 3ml×1瓶 11 封板膜 2张 6 显色剂B液 3ml×1/瓶 12 密封袋 1个 标本要求 1.标本采集后尽早进行提取,提取按相关文献进行,提取后应尽快进行实验。若不能马上进行试验,可将标本放于-20℃保存,但应避免反复冻融2.不能检测含NaN3的样品,因NaN3抑制辣根过氧化物酶的(HRP)活性。操作步骤1. 标准品的稀释:本试剂盒提供原倍标准品一支,用户可按照下列图表在小试管中进行稀释。80U/mL 5号标准品 150μl的原倍标准品加入150μl标准品稀释液 40U/mL 4号标准品 150μl的5号标准品加入150μl标准品稀释液 20 U/mL 3号标准品 150μl的4号标准品加入150μl标准品稀释液 10 U/mL 2号标准品 150μl的3号标准品加入150μl标准品稀释液 5 U/mL 1号标准品 150μl的2号标准品加入150μl标准品稀释液 2. 加样:分别设空白孔(空白对照孔不加样品及酶标试剂,其余各步操作相同)、标准孔、待测样品孔。在酶标包被板上标准品准确加样50μl,待测样品孔中先加样品稀释液40μl,然后再加待测样品10μl(样品最终稀释度为5倍)。加样将样品加于酶标板孔底部,尽量不触及孔壁,轻轻晃动混匀。3. 温育:用封板膜封板后置37℃温育30分钟。 4. 配液:将20倍浓缩洗涤液用蒸馏水20倍稀释后备用5. 洗涤:小心揭掉封板膜,弃去液体,甩干,每孔加满洗涤液,静置30秒后弃去,如此重复5次,拍干。6. 加酶:每孔加入酶标试剂50μl,空白孔除外。 7. 温育:操作同3。8. 洗涤:操作同5。9. 显色:每孔先加入显色剂A50μl,再加入显色剂B50μl,轻轻震荡混匀,37℃避光显色15分钟.10. 终止:每孔加终止液50μl,终止反应(此时蓝色立转黄色)。11. 测定:以空白空调零,450nm波长依序测量各孔的吸光度(OD值)。 测定应在加终止液后15分钟以内进行。操作程序总结: 计算 以标准物的浓度为横坐标,OD值为纵坐标,在坐标纸上绘出标准曲线,根据样品的OD值由标准曲线查出相应的浓度;再乘以稀释倍数;或用标准物的浓度与OD值计算出标准曲线的直线回归方程式,将样品的OD值代入方程式,计算出样品浓度,再乘以稀释倍数,即为样品的实际浓度。 注意事项1.试剂盒从冷藏环境中取出应在室温平衡15-30分钟后方可使用,酶标包被板开封后如未用完,板条应装入密封袋中保存。2.浓洗涤液可能会有结晶析出,稀释时可在水浴中加温助溶,洗涤时不影响结果。3.各步加样均应使用加样器,并经常校对其准确性,以避免试验误差。一次加样时间最好控制在5分钟内,如标本数量多,推荐使用排枪加样。4. 请每次测定的同时做标准曲线,最好做复孔。如标本中待测物质含量过高(样本OD值大于标准品孔第一孔的OD值),请先用样品稀释液稀释一定倍数(n倍)后再测定,计算时请最后乘以总稀释倍数(×n×5)。5. 封板膜只限一次性使用,以避免交叉污染。6.底物请避光保存。7.严格按照说明书的操作进行,试验结果判定必须以酶标仪读数为准.8.所有样品,洗涤液和各种废弃物都应按传染物处理。9.本试剂不同批号组分不得混用。10. 如与英文说明书有异,以英文说明书为准。保存条件及有效期1.试剂盒保存:;2-8℃。2.有效期:6个月
标准
2015.07.06
全体生物请注意,有癌症可以传染扩散!癌细胞是不会传染的,这是生物学的一个默认公知。不过这一常识已经在北美的东海岸被打破,一种致命的白血病样的病情已经从加拿大爱德华王子岛一路蔓延到纽约州。这可能通过在水中自由漂浮的癌细胞,肆意在软壳蛤中传染。美国哥伦比亚大学的Stephen Goff,形容这种癌症的广泛传播是“处于超级转移状态,能扩散到全新的动物”。这篇研究发表在本周Cell杂志。癌症,作为有机体的生命周期中的体细胞突变积累的结果,一般不会传染或传输给其他个体,受限于基于多态表面蛋白的免疫识别和拒绝,特别是在主要组织相容性复合体(MHC)的脊椎动物。不过一些肿瘤是受感染(如病毒)引起的。虽然这些感染源可以是传染性的,肿瘤仍然是在受感染的个体中通过体细胞的转化而形成。已知有两种情况下,肿瘤细胞本身作为传染性细胞可自然扩散传播:犬传染性性病肿瘤(CTVT)由性接触传播、塔斯马尼亚袋獾面部肿瘤病(DFTD)在个体之间通过传播。在这两种情况下,肿瘤显示出基因型不匹配它们的宿主——在所有受影响的动物中发现的肿瘤细胞是具有反映其原始主体的独特的基因型。
标准
2015.07.06
好消息!科学家开发出预防埃博拉病毒长效疫苗近日,来自英国普利茅斯大学的研究人员在国际学术期刊Vaccine发文宣称他们开发出针对埃博拉病毒的长效疫苗,应用该疫苗或可降低野生非洲猿的埃博拉病毒感染,对非洲埃博拉病毒的预防治疗具有重要意义。非洲猿是埃博拉病毒向人类传播的一个主要传染源。因此预防非洲猿发生埃博拉病毒感染能够降低埃博拉病毒在非洲人口中爆发的可能性。埃博拉病毒感染对非洲猿同样具有致死性,同时也是威胁这些野生种群生存的主要危险因素之一。针对埃博拉病毒的长效疫苗开发为稳定这些濒危猿类的种群数量以及保护人类免受埃博拉病毒感染提供了希望。研究人员在CMV病毒基础上开发了针对埃博拉病毒的长效疫苗。改造后的CMV病毒在去除了其免疫原性和种属特异性后,也能够比较容易得在个体之间进行传播,并且这一过程不会受到宿主CMV免疫的影响。因此基于CMV病毒这一特性开发的埃博拉病毒疫苗不需要直接接种便可在非洲猿身上诱导具有机体保护性的埃博拉病毒特异性免疫应答,并能够在野生猿个体之间广泛传播。应用该种疫苗,宿主产生的接种后免疫应答最少可维持接近四个月,长效免疫对于这种传播性疫苗方法取得最终成功具有至关重要的作用。
标准
2015.07.06
大鼠Toll样受体4(TLR4)酶联免疫分析试剂盒使用说明书本试剂盒仅供研究使用。检测范围: 96T1ng/mL - 32ng/mL使用目的:本试剂盒用于测定大鼠血清、血浆及相关液体样本中Toll 样受体4(TLR4)含量。实验原理本试剂盒应用双抗体夹心法测定标本中大鼠Toll 样受体4(TLR4)水平。用纯化的大鼠Toll 样受体4(TLR4)抗体包被微孔板,制成固相抗体,往包被单抗的微孔中依次加入Toll 样受体4(TLR4),再与HRP 标记的Toll 样受体4(TLR4)抗体结合,形成抗体-抗原-酶标抗体复合物,经过彻底洗涤后加底物TMB 显色。TMB 在HRP 酶的催化下转化成蓝色,并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的Toll 样受体4(TLR4)呈正相关。用酶标仪在450nm 波长下测定吸光度(OD 值),通过标准曲线计算样品中大鼠Toll 样受体4(TLR4)浓度。试剂盒组成1 30 倍浓缩洗涤液 20ml×1 瓶 7 终止液 6ml×1 瓶2 酶标试剂 6ml×1 瓶 8 标准品(64ng/mL) 0.5ml×1 瓶3 酶标包被板 12 孔×8 条 9 标准品稀释液 1.5ml×1 瓶4 样品稀释液 6ml×1 瓶 10 说明书 1 份5 显色剂A 液 6ml×1 瓶 11 封板膜 2 张6 显色剂B 液 6ml×1/瓶 12 密封袋 1 个标本要求1.标本采集后尽早进行提取,提取按相关文献进行,提取后应尽快进行实验。若不能马上进行试验,可将标本放于-20℃保存,但应避免反复冻融2.不能检测含NaN3 的样品,因NaN3 抑制辣根过氧化物酶的(HRP)活性。操作步骤1. 标准品的稀释:本试剂盒提供原倍标准品一支,用户可按照下列图表在小试管中进行稀释。32ng/mL 5 号标准品 150μl 的原倍标准品加入150μl 标准品稀释液16ng/mL 4 号标准品 150μl 的5 号标准品加入150μl 标准品稀释液8ng/mL 3 号标准品 150μl 的4 号标准品加入150μl 标准品稀释液4ng/mL 2 号标准品 150μl 的3 号标准品加入150μl 标准品稀释液2ng/mL 1 号标准品 150μl 的2 号标准品加入150μl 标准品稀释液2. 加样:分别设空白孔(空白对照孔不加样品及酶标试剂,其余各步操作相同)、标准孔、待测样品孔。在酶标包被板上标准品准确加样50μl,待测样品孔中先加样品稀释液40μl,然后再加待测样品10μl(样品最终稀释度为5 倍)。加样将样品加于酶标板孔底部,尽量不触及孔壁,轻轻晃动混匀。3. 温育:用封板膜封板后置37℃温育30 分钟。4. 配液:将30 倍浓缩洗涤液用蒸馏水30 倍稀释后备用5. 洗涤:小心揭掉封板膜,弃去液体,甩干,每孔加满洗涤液,静置30 秒后弃去,如此重复5 次,拍干。6. 加酶:每孔加入酶标试剂50μl,空白孔除外。7. 温育:操作同3。8. 洗涤:操作同5。9. 显色:每孔先加入显色剂A50μl,再加入显色剂B50μl,轻轻震荡混匀,37℃避光显色15 分钟.10. 终止:每孔加终止液50μl,终止反应(此时蓝色立转黄色)。11. 测定:以空白空调零,450nm 波长依序测量各孔的吸光度(OD 值)。 测定应在加终止液后15 分钟以内进行。操作程序总结:计算以标准物的浓度为横坐标,OD 值为纵坐标,在坐标纸上绘出标准曲线,根据样品的OD 值由标准曲线查出相应的浓度;再乘以稀释倍数;或用标准物的浓度与OD 值计算出标准曲线的直线回归方程式,将样品的OD 值代入方程式,计算出样品浓度,再乘以稀释倍数,即为样品的实际浓度。注意事项1.试剂盒从冷藏环境中取出应在室温平衡15-30 分钟后方可使用,酶标包被板开封后如未用完,板条应装入密封袋中保存。2.浓洗涤液可能会有结晶析出,稀释时可在水浴中加温助溶,洗涤时不影响结果。3.各步加样均应使用加样器,并经常校对其准确性,以避免试验误差。一次加样时间最好控制在5 分钟内,如标本数量多,推荐使用排枪加样。4. 请每次测定的同时做标准曲线,最好做复孔。如标本中待测物质含量过高(样本OD 值大于标准品孔第一孔的OD 值),请先用样品稀释液稀释一定倍数(n 倍)后再测定,计算时请最后乘以总稀释倍数(×n×5)。5. 封板膜只限一次性使用,以避免交叉污染。6.底物请避光保存。7.严格按照说明书的操作进行,试验结果判定必须以酶标仪读数为准.8.所有样品,洗涤液和各种废弃物都应按传染物处理。9.本试剂不同批号组分不得混用。10. 如与英文说明书有异,以英文说明书为准。保存条件及有效期1.试剂盒保存:;2-8℃。2.有效期:6 个月
标准
2015.07.03
大鼠P物质(SP)酶联免疫分析试剂盒使用说明书本试剂盒仅供研究使用。检测范围: 96T7ng/L -240ng/L 使用目的:本试剂盒用于测定大鼠血清、血浆及相关液体样本中P物质(SP)含量。实验原理本试剂盒应用双抗体夹心法测定标本中大鼠P物质(SP)水平。用纯化的大鼠P物质(SP)抗体包被微孔板,制成固相抗体,往包被单抗的微孔中依次加入P物质(SP),再与HRP标记的P物质(SP)抗体结合,形成抗体-抗原-酶标抗体复合物,经过彻底洗涤后加底物TMB显色。TMB在HRP酶的催化下转化成蓝色,并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的P物质(SP)呈正相关。用酶标仪在450nm波长下测定吸光度(OD值),通过标准曲线计算样品中大鼠P物质(SP)浓度。 试剂盒组成 1 30倍浓缩洗涤液 20ml×1瓶 7 终止液 6ml×1瓶 2 酶标试剂 6ml×1瓶 8 标准品(480ng/L) 0.5ml×1瓶 3 酶标包被板 12孔×8条 9 标准品稀释液 1.5ml×1瓶 4 样品稀释液 6ml×1瓶 10 说明书 1份 5 显色剂A液 6ml×1瓶 11 封板膜 2张 6 显色剂B液 6ml×1/瓶 12 密封袋 1个 标本要求 1.标本采集后尽早进行提取,提取按相关文献进行,提取后应尽快进行实验。若不能马上进行试验,可将标本放于-20℃保存,但应避免反复冻融2.不能检测含NaN3的样品,因NaN3抑制辣根过氧化物酶的(HRP)活性。 操作步骤1. 标准品的稀释:本试剂盒提供原倍标准品一支,用户可按照下列图表在小试管中进行稀释。240ng/L 5号标准品 150μl的原倍标准品加入150μl标准品稀释液 120 ng/L 4号标准品 150μl的5号标准品加入150μl标准品稀释液 60 ng/L 3号标准品 150μl的4号标准品加入150μl标准品稀释液 30 ng/L 2号标准品 150μl的3号标准品加入150μl标准品稀释液 15 ng/L 1号标准品 150μl的2号标准品加入150μl标准品稀释液 2. 加样:分别设空白孔(空白对照孔不加样品及酶标试剂,其余各步操作相同)、标准孔、待测样品孔。在酶标包被板上标准品准确加样50μl,待测样品孔中先加样品稀释液40μl,然后再加待测样品10μl(样品最终稀释度为5倍)。加样将样品加于酶标板孔底部,尽量不触及孔壁,轻轻晃动混匀。3. 温育:用封板膜封板后置37℃温育30分钟。 4. 配液:将30倍浓缩洗涤液用蒸馏水30倍稀释后备用。5. 洗涤:小心揭掉封板膜,弃去液体,甩干,每孔加满洗涤液,静置30秒后弃去,如此重复5次,拍干。6. 加酶:每孔加入酶标试剂50μl,空白孔除外。 7. 温育:操作同3。8. 洗涤:操作同5。9. 显色:每孔先加入显色剂A50μl,再加入显色剂B50μl,轻轻震荡混匀,37℃避光显色15分钟。10. 终止:每孔加终止液50μl,终止反应(此时蓝色立转黄色)。11. 测定:以空白空调零,450nm波长依序测量各孔的吸光度(OD值)。 测定应在加终止液后15分钟以内进行。 操作程序总结: 计算 以标准物的浓度为横坐标,OD值为纵坐标,在坐标纸上绘出标准曲线,根据样品的OD值由标准曲线查出相应的浓度;再乘以稀释倍数;或用标准物的浓度与OD值计算出标准曲线的直线回归方程式,将样品的OD值代入方程式,计算出样品浓度,再乘以稀释倍数,即为样品的实际浓度。 注意事项1.试剂盒从冷藏环境中取出应在室温平衡15-30分钟后方可使用,酶标包被板开封后如未用完,板条应装入密封袋中保存。2.浓洗涤液可能会有结晶析出,稀释时可在水浴中加温助溶,洗涤时不影响结果。3.各步加样均应使用加样器,并经常校对其准确性,以避免试验误差。一次加样时间最好控制在5分钟内,如标本数量多,推荐使用排枪加样。4. 请每次测定的同时做标准曲线,最好做复孔。如标本中待测物质含量过高(样本OD值大于标准品孔第一孔的OD值),请先用样品稀释液稀释一定倍数(n倍)后再测定,计算时请最后乘以总稀释倍数(×n×5)。5. 封板膜只限一次性使用,以避免交叉污染。6.底物请避光保存。7.严格按照说明书的操作进行,试验结果判定必须以酶标仪读数为准.8.所有样品,洗涤液和各种废弃物都应按传染物处理。9.本试剂不同批号组分不得混用。10. 如与英文说明书有异,以英文说明书为准。保存条件及有效期1.试剂盒保存:;2-8℃。2.有效期:6个月
标准
2015.07.03
肉眼读取DNA扩增结果我们都曾在化学课上根据pH试纸的颜色判断过溶液的酸碱度。NEB公司的研究团队以同样的理念(基于pH值的颜色改变)为基础,开发了一种对pH敏感的变色染料,给DNA扩增赋予了肉眼可见的颜色改变。这一技术在野外研究、田间试验、临床诊断等领域有着广阔的应用前景。
标准
2015.07.03
大鼠(Rat)肿瘤坏死因子-α(TNF-α) ELISA 检测试剂盒本试剂仅供研究使用 标本:血清或血浆 试验原理:TNF- α试剂盒是固相夹心法酶联免疫吸附实验(ELISA).已知TNF- α浓度的标准品、未知浓度的样品加入微孔酶标板内进行检测。先将TNF- α和生物素标记的抗体同时温育。洗涤后,加入亲和素标记过的HRP。再经过温育和洗涤,去除未结合的酶结合物,然后加入底物A、B,和酶结合物同时作用。产生颜色。颜色的深浅和样品中TNF- α的浓度呈比例关系。 试剂盒内容及其配制试剂盒成份(2-8℃保存) 96孔配置 48孔配置 配制 96/48人份酶标板 1块板(96T) 半块板(48T) 即用型 塑料膜板盖 1块 半块 即用型 标准品:400pg/ml 1瓶(0.6ml) 1瓶(0.3ml) 按说明书进行稀稀 空转移趋化生长因子β1对照 1瓶(1.0ml) 1瓶(0.5ml) 即用型 标准品稀释缓冲液 1瓶(5ml) 1瓶(2.5ml) 即用型 生物素标记的抗TNF- α抗体 1瓶(6ml) 1瓶(3.0ml) 即用型 亲和链酶素-HRP 1瓶(10ml) 1瓶(5.0ml) 即用型 洗涤缓冲液 1瓶(20ml) 1瓶(10ml) 按说明书进行稀释 底物A 1瓶(6.0ml) 1瓶(3.0ml) 即用型 底物B 1瓶(6.0ml) 1瓶(3.0ml) 即用型 终止液 1瓶(6.0ml) 1瓶(3.0ml) 即用型 标本稀释液 1瓶(12ml) 1瓶(6.0ml) 即用型 自备材料1. 蒸馏水。2. 加样器:5ul、10ul、50ul、100ul、200ul、500ul、1000ul。3. 振荡器及磁力搅拌器等。 安全性1. 避免直接接触终止液和底物A、B。一旦接触到这些液体,请尽快用水冲洗。2. 实验中不要吃喝、抽烟或使用化妆品。3. 不要用嘴吸取试剂盒里的任何成份。 操作注意事项1. 试剂应按标签说明书储存,使用前恢复到室温。稀稀过后的标准品应丢弃,不可保存。2. 实验中不用的板条应立即放回包装袋中,密封保存,以免变质。3. 不用的其它试剂应包装好或盖好。不同批号的试剂不要混用。保质前使用。4. 使用一次性的吸头以免交叉污染,吸取终止液和底物A、B液时,避免使用带金属部分的加样器。5. 使用干净的塑料容器配置洗涤液。使用前充分混匀试剂盒里的各种成份及样品。6. 洗涤酶标板时应充分拍干,不要将吸水纸直接放入酶标反应孔中吸水。7. 底物A应挥发,避免长时间打开盖子。底物B对光敏感,避免长时间暴露于光下。避免用手接触,有毒。实验完成后应立即读取OD值。8. 加入试剂的顺序应一致,以保证所有反应板孔温育的时间一样。9. 按照说明书中标明的时间、加液的量及顺序进行温育操作。 样品收集、处理及保存方法1、 血清-----操作过程中避免任何细胞刺激。使用不含热原和内毒素的试管。收集血液后,1000×g离心10分钟将血清和红细胞迅速小心地分离。2、 血浆-----EDTA、柠檬酸盐、肝素血浆可用于检测。1000×g离心30分钟去除颗粒。3、 细胞上清液---1000×g离心10分钟去除颗粒和聚合物。4、 组织匀浆-----将组织加入适量生理盐水捣碎。1000×g离心10分钟,取上清液5、 保存------如果样品不立即使用,应将其分成小部分-70 ℃保存,避免反复冷冻。尽可能的不要使用溶血或高血脂血。如果血清中大量颗粒,检测前先离心或过滤。不要在37℃或更高的温度加热解冻。应在室温下解冻并确保样品均匀地充分解冻。 试剂的准备1. 标准品:标准品的系列稀释应在实验时准备,不能储存。稀释前将标准品振荡混匀。稀释比例按下表中进行:400 pg/ml (6号标准品) 原倍浓度不用稀释直接加入50ul。 200 pg/ml (5号标准品) 100ul的原倍标准品加入100ul的标准品稀释液 100 pg/ml (4号标准品) 100ul的5号标准品加入100ul的标准品稀释液 50 pg/ml (3号标准品) 100ul的4号标准品加入100ul的标准品稀释液 25 pg/ml (2号标准品) 100ul的3号标准品加入100ul的标准品稀释液 12.5 pg/ml (1号标准品) 100ul的2号标准品加入100ul的标准品稀释液 0 pg/ml (空转移趋化生长因子β1对照) 原始浓度不用稀释直接加入50ul。 2. 洗涤缓冲液(50×)的稀释:蒸馏水50倍稀释。 操作步骤1. 使用前,将所有试剂充分混匀。不要使液体产生大量的泡沫,以免加样时加入大量的气泡,产生加样上的误差。2. 根据待测样品数量加上标准品的数量决定所需的板条数。每个标准品和空转移趋化生长因子β1孔建议做复孔。每个样品根据自己的数量来定,能使用复孔的尽量做复孔。标本用标本稀释液1:1稀释后加入50ul于反应孔内。3. 加入稀释好后的标准品50ul于反应孔、加入待测样品50ul于反应孔内。立即加入50ul的生物素标记的抗体。盖上膜板,轻轻振荡混匀,37℃温育1小时。4. 甩去孔内液体,每孔加满洗涤液,振荡30秒,甩去洗涤液,用吸水纸拍干。重复此操作3次。如果用洗板机洗涤,洗涤次数增加一次。5. 每孔加入80ul的亲和链酶素-HRP,轻轻振荡混匀,37℃温育30分钟。6. 甩去孔内液体,每孔加满洗涤液,振荡30秒,甩去洗涤液,用吸水纸拍干。重复此操作3次。如果用洗板机洗涤,洗涤次数增加一次。7. 每孔加入底物A、B各50ul,轻轻振荡混匀,37℃温育10分钟。避免光照。8. 取出酶标板,迅速加入50ul终止液,加入终止液后应立即测定结果。9. 在450nm波长处测定各孔的OD值。 建议使用的实验方案标准品浓度(pg/ml) A 400 400 样品 样品 样品 样品 样品 样品 样品 样品 样品 样品 B 200 200 样品 样品 样品 样品 样品 样品 样品 样品 样品 样品 C 100 100 样品 样品 样品 样品 样品 样品 样品 样品 样品 样品 D 50 50 样品 样品 样品 样品 样品 样品 样品 样品 样品 样品 E 25 25 样品 样品 样品 样品 样品 样品 样品 样品 样品 样品 F 12.5 12.5 样品 样品 样品 样品 样品 样品 样品 样品 样品 样品 G 0 0 样品 样品 样品 样品 样品 样品 样品 样品 样品 样品 H 样品 样品 样品 样品 样品 样品 样品 样品 样品 样品 样品 样品 局限6号标准品以上的结果为非线性的,根据此标准曲线无法得到精确的结果。 试剂盒性能1. 灵敏度:最小的检测浓度小于1号标准品。稀释度的线性。样品线性回归与预期浓度相关系数R值为0.990。2. 特异性:不与其它细胞因子反应。3. 重复性:板内、板间变异系数均小于10%。 结 果 判 断 与 分 析1、仪器值:于波长450nm的酶标仪上读取各孔的OD值 2、以吸光度OD值为纵坐标(Y),相应的TNF- α标准品浓度为横坐标(X),做得相应的曲线,样品的TNF- α含量可根据其OD值由标准曲线换算出相应的浓度。 3、检测值范围:0-400pg/ml 4、敏感度: 1.0 pg/ml
标准
2015.07.01
淋巴管的胚胎起源淋巴内皮过去被认为完全是从静脉内皮的转分化形成的。本期Nature上发表的两篇研究论文显示,这种脉管的起源要比人们所预料的更为多样。Karina Yaniv及同事通过对转基因斑马鱼胚胎进行活体成像来跟踪各个淋巴内皮细胞和它们后代的世系。他们发现,淋巴祖细胞是从以前没有被表征的位于主静脉床上的一组多能成血管细胞形成的,后者具有不仅形成淋巴细胞、而且形成动脉细胞和静脉细胞的潜力。Paul Riley及同事通过对小鼠采用“genetic fate-mapping”方法显示,心脏淋巴内皮细胞有两个起源,既涉及静脉内皮细胞,又涉及源自卵黄囊细胞的另一个非静脉祖细胞来源。他们还发现心脏中的淋巴管生成会受到心肌梗塞的影响,并且还提出,通过VEGF-C促进淋巴管生成可以在发生梗塞后改善心脏功能。
标准
2015.07.01
正面记忆的力量回想正面记忆是否能减轻抑郁?Susumu Tonegawa 及同事利用与一个正面的、中性的或负面的经历相关的、通过光遗传学方法标记的特定海马体记忆印迹对这一问题进行了研究。这些记忆之后可以通过光被人工激活。正面印迹的急性激活被发现会抑制暴露于慢性压力的小鼠类似抑郁症的行为,这是由 “海马体-杏仁核-伏隔核”通道介导的一个效应。重要的是,正面印迹的慢性激活也会抑制处于压力状态下的小鼠类似抑郁症的行为,即便在激活已经结束以后也是这样,说明这种抗抑郁效应并不取决于对印迹的实时人工激活。作者提出,对与一个正面记忆相关的海马体齿状回细胞的直接激活,也许能为减轻一类与抑郁相关的行为提供一个潜在的治疗节点,尽管在目前阶段还不清楚这些发现对人类是否适用。
标准
2015.07.01
Goat Anti-Rat Toll-like receptor 4 Storage: 2 - 8 °C Package size: 96 determinations PRINCIPLE OF THE METHODThe TLR-4 kit is a solid phase phase sandwich enzyme linked immuno sorbent assay(ELISA). Samples , including standards of known TLR-4 concentrations and unknowns are pipetted into these wells. During the first incubation, the TLR-4 antigen and a biotinylated monoclonal antibody specific for TLR-4 are simultaneously incubated. After washing, the enzyme(streptavidin-peroxydase)is added. After incubation and washing to remove all the unbound enzyme, a substrate solution which is acting on the bound enzyme is added to induce a coloured reaction product. The intensity of this coloured product is directly proportional to the concentration of TLR-4 present in the samples. REAGENTS PROVIDED AND RECONSTTTUTIONREAGENTS(Store at 2-8℃) 1×96 WELLS 0.5×96 WELLS RECONSTTTUTION 96/48-wells microtiter plates 1 0.5 Ready-to-use Plastiv cover 2 1 Ready-to-use Standard: 8.0ng/ml 1Vials (0.6ml) 0.5Vials (0.3ml) See reagents preparation on page 3 Blank control 1Vials (1.0ml) 1Vials (0.5ml) Ready-to-use Standard Diluent 1Vials (4.0ml) 1Vials (2.0ml) Ready-to-use Biotinylated anti-TLR-4 1Vials (6.0ml) 1Vials (3.0ml) Ready-to-use Streptavidin-HRP 1Vials (8.0ml) 1Vials (4.0ml) Ready-to-use Washing Buffer 1Vials (20ml) 1Vials (10ml) 50× concentrate Substrate A 1Vials (6.0ml) 1Vials (3.0ml) Ready-to-use Substrate B 1Vials (6.0ml) 1Vials (3.0ml) Ready-to-use Stopping Solution 1Vials (6.0ml) 1Vials (3.0ml) Ready-to-use Sample Diluent 1Vials (12ml) 1Vials (6.0ml) Ready-to-use MATERIAL REQUIRED BUT NOT PROVIDEDl Distilled waterl Pipettes:10ul、50ul、100ul、200ul、1000ul。l Vortex mixer and magnetic stirrer. SAFETYl For research use onlyl Avoid any skin contact with H2SO4 and TMB. In case of contact, wash thoroughly water.l Do not eat, drink, smoke or apply cosmetics where kit reagents are used.l Do not pipette by mouth. PROCEDURAL NOTES/LAB.QUALITY CONTROLl When not in use, kit components should be stored refrigerated or frozen as indicated on vials or bottles. All reagents should be warmed to room temperature before use. Lyophilized standards should be discarded after use.l Once the desired number of strips has been removed, immediately reseal the bag to protect the remaining strips from edterioration.l Cover or cap all reagents when not in use.l Do not mis or interchange reagents between different lots.l Do not use reagents beyond the expiration date of the kit .l Use a clean disposable plastic pipette tip for each reagent, standard, or specimen addition in order to avoid cross-contamination, for the dispensing of H2SO4 and substrate solution, avoid pipettes with metal parts.l Use a clean plastic container to prepare the washing solution.l Thoroughly mix the reagents and samples before use by agitation or swirling.l All residual washing liquid must be drained from the wells by efficient aspiration or by decantation followed by tapping the plate forcefully on absorbent paper. Never insert absorbent paper directly into the wells.l The TMB solution is light sensitive. Avoid prolonged exposure to light, also, avoid contact of the TMB solution with metal to prevent colour development. Warning TMB is toxic avoid direct contact with hands. Dispose off properly. If a dark blue colour develops within a few minutes after preparation, this indicates that the TMB solution has been contaminated and must be discarede. Read absorbances within 1 hour after completion of the assay.l When pipetting reagents, maintain a consistent order of addition from well-to-well. This will ensure equal incubation times for all wells.l Respect incubation times described in the assay procedure. SPECIMEN COLLECTION\ PROCESSING AND STORAGEl Serum---Avoid any inintentional stimulation of the cells by the procedure. Use pyrogen\endotoxin free collecting tubes. Serum should be removed rapidly and carefully from the red cells after clothing. For that, after clothing, centrifuge at approximately 1000×g for 10 min and remove serum.l Plasma---EDTA\ citrate and heparin plasma can be assayed. Spin samples at 1000×g for 30 min remove particulates. Harvest plasma.l Cell culture supernatants---Remove particulates and aggregates by spinning at approximately 1000×g for 10 min.l Storage---If not analyzed shortly after collection, samples should be aliquoted(250-500ul) to avoid freeze-thaw cycles and stored frozen at -70℃. Avoid multiple freeze-thaw cycles of frozen specimens. When possible, avoid use of badly hemolyzed or lipemic sera. If large amounts of particles are present, this should be removed prior to assay by centrifugation or filtration.l Recommendation---Do not thaw by heating at 37℃ or 56℃. Thaw at room temperature and make sure that sample is completely thawed and homogenous before assaying. PREPARATION OF REAGENTSl Standards: Standard have to be reconstituled with the volume of standard buffer diluent indicated on the vial. This reconstitution produces a stock solution of 8.0ng/ml TLR-4. Allow standard to stand for 5 l minutes with gentle swirling prior to making dilutions. Serial dilutions of standard must be made before each assys and cannot be stored. 8.0 ng/ml (6 Standard) Original density 50ul。 4.0 ng/ml (5 Standard) 100ul 6 Standard +100ul diludent 2.0 ng/ml (4 Standard) 100ul 5 Standard +100ul diludent 1.0 ng/ml (3 Standard) 100ul 4 Standard +100ul diludent 0.5 ng/ml (2 Standard) 100ul 3 Standard +100ul diludent 0.25 ng/ml (1 Standard) 100ul 2 Standard +100ul diludent 0 ng/ml Blank Control 50ul。 l Washing buffer 50×concentrate: Dilute 50 times in distilled water. ASSAY METHODl Before use, mix all reagents thoroughly without making foam.l Determine the number of microwell strips required to test the desired number of samples,plus appropriate number of wells needed for running blanks standards. Each sample, standard and blank should be assayed in duplicate. Remove sufficient microwell strips from the pouch.l Add 50ul of standard diluent to standard wells B1,B2, C1,C2, D1,D2, E1, E2, F1,F2. Reconstitute standard vial with the appropriate volume as described in the chapter reagents preparation. Preparation. Pipet 100ul of standard into wells A1 and A2 (see plate scheme below). Transfer 50ul from A1 and A2 to B1 and B2 wells. Mix the contents by repeated aspirations and ejections. Take care not to scratch the inner surface of microwells. Repat this procedure from the wells B1,B2 to wells C1,C2 and from wells C1,C2 to D1,D2 and so on creating two parallel rows of TLR-4 standard dilutions ranging,Add 50ul of standard diluent to the bland wells.l Dilute samples 1:1 distribing 50ul of sample into 50ul of dilluent ,Add 50ul of diluted sample to wells..l Add 50ul of diluted biotinylated anti-TLR-4 to all wells.l Cover with a plate vover and incubate for 1 hour at 37℃.l Remove the cover and wash the plate as follows: ⑴ aspirate the liquid from each well, ⑵ dispensse 0.3ml of washing solution into each well. ⑶ Aspirate again the contet of each well after 0.5 minute. ⑷ Repeat steps ⑵ and ⑶ three times.l Distribute 60ul of streptavidin-HRP solution to all wells, including blank wells.l Cover and incubate 30 min at 37℃.l Remove the cover and empty wells, Wash microwell strips according to step, Proceed immediately to the next step.l Add 50ul Substrate A and Substrate B to each well。Incubate for 10 min at 37℃。l The enzyme-substrate reaction is stopped by quickly pipetting 50ul of H2SO4. stop reagent into each well, including the blank wells, to completely and uniformly inactivate the enzyme. Results must be red immediately after the addition of H2SO4.l Read absorbance of each well on a spectrophotometer using 450nm as the primary wavelength and optionally 620nm (610nm to 650nm is acceptable ) as the reference wavelength. SUGGESTED PLATE SCHEMEStandardconcentrations(ng/ml) A 8.0 8.0 sample sample sample sample sample sample sample sample sample sample B 4.0 4.0 sample sample sample sample sample sample sample sample sample sample C 2.0 2.0 sample sample sample sample sample sample sample sample sample sample D 1.0 1.0 sample sample sample sample sample sample sample sample sample sample E 0.5 0.5 sample sample sample sample sample sample sample sample sample sample F 0.25 0.25 sample sample sample sample sample sample sample sample sample sample G 0 0 sample sample sample sample sample sample sample sample sample sample H sample sample sample sample sample sample sample sample sample sample sample sample LIMITATIONS OF THE PROCEDURE Do not extrapolate the standard curve beyond the max standard curve point. The dose-response is non-linear in this region and good accruacy is difficult to obtain. CALCULATION OF RESULTS The minimum detectable concentration in this assay is estimated to be 0.01ng/ml
标准
2015.06.29
欧洲最大生物医学研究中心在争议中启航15年前,Paul Nurse有一个他现在称为“愚蠢的主意”。这位曾获得诺贝尔奖的遗传学家提议将英国伦敦两家最大的生物医学研究中心合并在一起。他建议在位于伦敦南部格林威治半岛的千禧巨蛋(周长1千米、高52米的开放式展览中心)里建立一个巨型实验室。“这是疯狂的,因为这里不是作为一个研究所而建设的。它收效甚微。”实际上,Nurse的主意在别处有了收获:距离伦敦市中心西北约10公里的地方。今年11月,他将打开弗朗西斯·克里克研究所的大门。这家研究所甚至比他最初的预想更野心勃勃。2021年,这家耗资7亿英镑、占地9.3万平方米的研究所将全功率运行,届时将有1600位科学家和工作人员在这里工作,它将成为欧洲最大的单一生物医学实验室。Nurse和英国政府将克里克研究所标榜为给予英国科学的馈赠:它将作出开创性的发现、吸引全世界无数优秀年轻科学家来到英国,并促进英国生物产业的繁荣。“这里将会相当不同。我觉得不是每个人都意识到这里都放了些什么。”Nurse说。如果能够奏效,这个有些“古怪”的科学基础设施将指导其他国家在未来建设自己的实验室。但也有人质疑克里克研究所是否值得冒险。他们警告称,该中心将会成为一个吞噬公共和私人研究经费的黑洞,并将扩大英国科学的贫富差距。“这是一只投资巨鳄。它必须成功,不然我们还能做什么?”英国萨塞克斯大学科学政策专家James Wilsdon说。雄心壮志可以说,克里克研究所的诞生是必然的。到上世纪90年代末,英国两家最受欢迎的生物医学研究所需要找个新家。国家医学研究所(NIMR)坐落在伦敦北部一个二战前的建筑里,设施已经老化。这里的一些设备由慈善机构皇家癌症研究基金(后更名为英国癌症研究中心——CRUK)资助。当提议建立千禧巨蛋巨型实验室时,Nurse正担任该中心主任。2003年,Nurse离开英国出任美国洛克菲勒大学校长。但他并没有放弃自己的“疯狂”想法。2006年,一份极具影响力的政府评估(Cooksey报告)认为,该国“存在未能收获公共在健康研究领域投资应产生的全部经济、健康和社会福利的风险”,并且呼吁增加资助者之间的合作和更大的生物医学研究创新。因此,时任NIMR所长Keith Peters评估了Nurse合并两家研究机构的建议。Peters选中了伦敦北部的一块地,位于大不列颠图书馆和圣潘克拉斯火车站之间。为了支付新中心的相关费用,他把附近的伦敦大学学院和维康信托拉了进来;之后伦敦大学国王学院和帝国理工学院也加入,每家机构捐助了400万英镑用于支付建造费用。这些大学也会为克里克研究所的研究人员提供设施,尤其是物理学家和工程师,并允许该机构使用它们的附属医院。克里克研究所的资助者们希望看到这里的成果走向临床。研究人员涉及从干细胞到流行病的各个领域,他们将能使用药物筛检机器人、高性能显微镜和超过20万只动物,大多数是老鼠和斑马鱼。该研究所将在最初几年迅速发展。NIMR 和CRUK的伦敦研究所(LRI)的90多个团队领导将成为克里克研究所的首批入驻者,并将于明年年初入驻新机构。另外约120个团队领导职位将由新招募的年轻研究人员占据。来自NIMR和LRI的已经获得永久职位的研究人员还将保留该职位,但当他们退休后,享有终身职位的团队领导将缩减2/3。Nurse表示,员工流动率将让克里克研究所遵循干细胞或基因组编辑等最新的研究趋势。“不断振兴革新的研究所能避免过于保守和僵化。”摸着石头过河“温和的无序”是Nurse对自己所希望的克里克研究所未来的描述。这里的研究人员不属于任何学部或部门,他们在超级实验室中的办公位置取决于他们使用的核心设备是什么。在Nurse的愿景中,研究人员将形成针对微生物学等的基层兴趣小组。“大多数人认为这是疯狂的,但我认为这令人非常兴奋和激动,因为这里的组织结构将由下往上。”Nurse说。诺奖得主、科罗拉多大学生物化学家、霍华德·休斯医学研究所(HHMI)前所长Tom Cech表示,其他国家的科学资助者将密切关注克里克研究所。他也有一些不同寻常的冒险经历。在Cech的任期内,HHMI开设了生物科学研究所珍利亚农场研究园区,该园区团队规模很小,取消了终身职位,并强调跨学科研究。“如果没有人做这些实验,那么问题仍然处于假设状态。像珍利亚农场和克里克研究所这样的实验,对社会很重要。”Cech说,因为它们能够激发作研究的新方式。克里克研究所的支持者还希望该机构能通过架设实验室和市场间的桥梁而成为英国经济引擎。Nurse的副手、首席运行官David Roblin曾在医药巨头辉瑞担任研发主管,并也曾在小型生物技术公司供职。“虽然我是一个产业科学家,但我并不会让它成为一个制药公司。”Roblin说,“克里克研究所是一个发现型研究所,并对产业转化非常感兴趣。”Roblin正试着吸引制药公司让研究人员入驻克里克研究所,以便加速成果的临床转化率。但与私人公司不同,这里的所有发现都将公开报道。而且知识专利也将属于克里克研究所。即便在一个充斥着玻璃和钢筋超级建筑的城市里,克里克研究所也将是一个壮观的地标性建筑。1999年被授予爵位的Nurse表示,他的员工有时称这里为“Paul爵士的大教堂”。该研究所的内部令人感觉十分巨大。为了方便人们定向,不同楼层的墙壁被刷上了不同的颜色,并且还开发了智能手机App。“我想我们是蓝色的,但我不记得了。”一位将于明年搬到这里的科学家说。这里的4层地下楼层将放置电子显微镜等敏感设备。与其他许多新实验室一样,克里克研究所也鼓励研究人员间的互动。每个地上楼层研究中心周围布满了会议室、咖啡区和“协同空间”。“这是我感到最兴奋的事情之一。”从NIMR 搬到这里的发育生物学家James Briscoe说。但也有人认为免疫学家与物理学家喝咖啡并没有必要。而克里克研究所的规模则是另一个症结所在。“每个人都相信科学变得更大且资金更密集。”曼彻斯特大学科学与技术政策专家Flanagan说,“但有一些证据表明,小群体比大鳄更有效率。”连锁反应Nurse表示,克里克研究所将作为英国的科学孵化器:他希望那些离开这里的研究人员也能为该国大学贡献自己的聪明才智。“我半开玩笑地说,他们应该爱上住在这里的人。”他说。但批评者表示,Nurse在年轻的伦敦人面临的严峻经济挑战方面表现得油嘴滑舌。这里的博士后甚至首席研究员都无法在研究所附近买一间公寓。而且他们怀疑,克里克研究所是否将成为Nurse和政府认为的那种孵化器。“它可能会这样,但没有事实上的理由证明它为何将这样。”布里斯托大学细胞生物学家David Stephens说。还有人担忧克里克研究所将变得“大而不倒”——它的公私资助者会将其置于其他优先事项的前面。除了7亿英镑的建设成本,该研究所将有每年1.5亿英镑的年度预算,这些钱将出自政府的医学研究委员会(MRC)、CRUK和维康信托。而一个平稳甚至缩水的政府科学预算也会放大这些担忧。“一个研究所分掉一大部分钱会让其他人更难吸引经费。”Stephens说。但Nurse表示,这些投诉是没有理由的“恐怖故事”,出自那些担忧自己经费的科学家之手。他认为克里克研究所的规模能帮助它进行世界级研究以及确保经费支持。但现实会怎样?评估克里克研究所最终会成功或失败将十分困难。很快将进入研究所的研究人员已经是科学领军人物,并将继续进行高水平工作。但这并不够,Cech表示,克里克研究所是否成功,应由它能否作出大学研究人员用同样经费难以作出的成果来判断。而Nurse也承认克里克研究所的结构可能无法达到其崇高目标。“我认为我们有合理的机会。”他说。但他也愿意改变。“如果我们缺乏部门会带来混乱,那么我会举白旗,并说‘好,我们将做其他对的事情’。”他说,“我不是偏执狂。
商机
2015.06.29
Rat trypsin FOR RESEARCH USE ONLY Assay range:0.6ng/mL - 16ng/mL 96 determinationsPurposeThis kit allows for the determination of trypsin concentrations in Rat serum, cell culture supernates and other biological fluids Principle of the assayThe kit assay Rat trypsin level in the sample,use Purified Rat trypsin antibody to coat microtiter plate wells, make solid-phase antibody, then add trypsin to wells, Combined trypsin antibody which With HRP labeled,become antibody - antigen - enzyme-antibody complex, after washing Completely, Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of Rat trypsin in the samples is then determined by comparing the O.D. of the samples to the standard curve.Materials provided with the kit1 wash solution 20ml×1bottle 7 Stopp Solution 6ml×1 bottle 2 HRP-Conjugate reagent 6ml×1 bottle 8 Standard(32ng/mL) 0.5ml×1 bottle 3 Microelisa stripplate 12well×8strips 9 Standard diluent 1.5ml×1bottle 4 Sample diluent 6ml×1 bottle 10 Instruction 1 5 Chromogen Solution A 6ml×1 bottle 11 Closure plate membrane 2 6 Chromogen Solution B 6ml×1 bottle 12 Sealed bags 1 Specimen requirements1. extract as soon as possible after Specimen collection,and according to the relevant literature, and should be experiment as soon as possible after the extraction. If it can’t, specimen can be kept in -20 ℃ to preserve, Avoid repeated freeze-thaw cycles.2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.Assay procedure1. Dilute and add sample:Dilute Original density Standard as follow table:16ng/mL 5 Standard 150μl Original density Standard+150μl Standard diluent 8ng/mL 4 Standard 150μl 5 Standard+150μl Standard diluent 4ng/mL 3 Standard 150μl 4 Standard+150μl Standard diluent 2ng/mL 2 Standard 150μl 3 Standard +150μl Standard diluent 1ng/mL 1 Standard 150μl 2 Standard +150μl Standard diluent 2.add sample:Set blank wells separately (blank comparison wells don’t add sample and HRP-Conjugate reagent, other each step operation is same). testing sample well. add Sample dilution 40μl to testing sample well, then add testing sample 10μl (sample final dilution is 5-fold), add sample to wells , don’t touch the well wall as far as possible, and Gently mix.3.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37℃.4.Configurate liquid: 30-fold(or 20-fold) wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.5.washing:Uncover Closure plate membrane, discard Liquid, dry by swing, add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by pat.6.add enzyme:Add HRP-Conjugate reagent 50μl to each well, except blank well. 7.incubate:Operation with 3.8.washing:Operation with 5.9.color:Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 37℃10.Stop the reaction:Add Stop Solution50μl to each well, Stop the reaction(the blue color change to yellow color).11.assay:take blank well as zero , Read absorbance at 450nm after Adding Stop Solution and within 15min.Steps descriptionStandard, Sample diluent Add Standard, Sample diluent, incubate for 30 min at 37℃. Wash 5 time,Add HRP-Conjugate reagent, incubate for 30 min at 37℃. Wash 5 times,Add Chromogen Solution A and B, incubate for 30 min at 37℃. Add Stopp Solution Read absorbance at 450nm within 15 min calculate CalculateTake the standard density as the horizontal, the OD value for the vertical ,draw the standard curve on graph paper, Find out the corresponding density according to the sample OD value by the Sample curve, multiplied by the dilution multiple, or calculate the straight line regression equation of the standard curve with the standard density and the OD value ,with the sample OD value in the equation, calculate the sample density, multiplied by the dilution factor, the result is the sample actual density.Important notes1. The kit takes out from the refrigeration environment should be balanced 15-30 minutes in the room temperature, ELISA plates coated if has not use up after opened, the plate should be stored in Sealed bag.2. washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result.3. add Sample with sampler Each step, And proofread its accuracy frequently, avoids the experimental error. add sample within 5 min, if the number of sample is much , recommend to use Volley .4. if the testing material content is excessively higher (The sample OD is bigger than the first standard well ),please dilute Sample (n-fold), Please diluente and multiplied by the dilution factor.(×n×5).5. Closure plate membrane only limits the disposable use, to avoid cross-contamination.6. The substrate evade the light preservation.7. Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.8. All samples, washing buffer and each kind of reject should according to infective material process.9. Do not mix reagents with those from other lots. Storage and validity1.Storage: 2-8℃.2.validity: six months
标准
2015.06.25