搜全站
搜展位
武汉费斯德生物科技有限公司
关注
已关注
已认证
粉丝量 0
产品介绍
其它参数
应用：Pull-Down
Rap1 Pull-Down Activation Assay Kit
Cat. # 81401
Introduction
A. Background
Small GTPases are a super-family of cellular signaling regulators. The small Ras-like GTPase Rap1 is an evolutionary conserved protein that originally gained interest because of its capacity to revert the morphological phenotype of Ras-transformed fibroblasts. Rap1 is regulated by a large number of stimuli that include growth factors and cytokines, but also physical force and osmotic stress. Rap1 was shown to regulate multiple basic cellular processes. The best studied aspect of Rap1 function in endothelial cells involved its role in regulation of cell-cell junction formation and remodeling.
Rap1 Activation Assay Kit is based on the configuration-specific monoclonal antibody that specifically recognizes Rap1-GTP, but not Rap1-GDP. Given the high affinity of monoclonal antibodies to their antigens, the activation assay could be performed in a short time. This assay provides the reliable results with consistent reproducibility.
The anti-Rap1-GTP monoclonal antibody can also be used to monitor the activation of Rap1 in cells and in tissues by immunohistochemistry.
B. Assay Principle
The Rap1 Activation Assay Kit uses configuration-specific anti-Rap1-GTP Mouse monoclonal antibody to measure Rap1-GTP levels in cell extracts or in vitro GTPγS loading Rap1 activation assays. Anti-Rap1-GTP mouse monoclonal antibody is first incubated with cell lysates containing Rap1-GTP. Next, the GTP-bound Rap1 is pulled down by protein A/G agarose. Finally, the precipitated Rap1-GTP is detected through immunoblot analysis using Anti-Rap1 Rabbit Polyclonal Antibody.
C. Kit Components
1. Anti-Rap1-GTP Mouse Monoclonal Antibody (Cat. # 26912): One vial – 35 µL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibody specifically recognizes Rap1-GTP from all vertebrates.
2. Protein A/G Agarose (Cat. # 30301): One vial – 600 µL of 50% slurry.
3. 5X Assay/Lysis Buffer (Cat. # 30302): One bottle – 30 mL of 250 mM Tris-HCl, pH 8, 750 mM NaCl, 50 mM MgCl2, 5 mM EDTA, 5% Triton X-100.
4. Anti-Rap1 Rabbit Polyclonal Antibody (Cat. # 21156): One vial – 50 µL (1 mg/mL) in PBS, pH 7.4, contained 50% glycerol.
5. 100X GTPγS (Cat. # 30303): One vial – 50 µl at 10 mM, use 5 µL of GTPγS for GTP-labeling of 0.5 mL of cell lysate.
6. 100X GDP (Cat. # 30304): One vial – 50 µl at 100 mM, use 5 µL of GDP for GDP-labeling of 0.5 mL of cell lysate.
7. HRP-Goat Anti-Rabbit IgG (Cat. #29002): 50 µL (0.4 mg/mL) in PBS, pH 7.4, contained 50% glycerol.
D. Materials Needed but Not Supplied
1. Stimulated and non-stimulated cell lysates
2. Protease inhibitors
3. 4 °C tube rocker or shaker
4. 0.5 M EDTA at pH 8.0
5. 1.0 M MgCl2
6. 2X reducing SDS-PAGE sample buffer
7. Electrophoresis and immunoblotting systems
8. Immunoblotting wash buffer such as TBST (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.05% Tween-20)
9. Immunoblotting blocking buffer (TBST containing 5% Non-fat Dry Milk or 3% BSA)
10. ECL Detection Reagents
E. Example Results
The following figure demonstrates example results seen with the Rap1 Activation Assay Kit. For reference only.
Rap1 Activation Assay. Purified Rap1 proteins were immunoprecipitated after treated with GDP (lane 1) or GTPγS (lane 2). Immunoprecipitation was done with the anti-Rap1-GTP monoclonal antibody (Cat. No. 26912). Immunoblot was with an anti-Rap1 polyclonal antibody (Cat. # 21156).
Assay Procedure
A. Reagent Preparation
1X Assay/Lysis Buffer: Mix the 5X Stock (Cat. # 30301) briefly and dilute to 1X in deionized water. Just prior to usage, add protease inhibitors such as 1 mM PMSF, 10 µg/mL leupeptin, or 10 µg/mL aprotinin.
B. Sample Preparation
Adherent Cells
1. Culture cells (one 10-cm plate, ~107 cells) to approximately 80-90% confluence. Stimulate the cells with activator or inhibitor as desired.
2. Aspirate the culture media and wash twice with ice-cold PBS.
3. Completely remove the final PBS wash and add ice-cold 1X Assay/Lysis Buffer (See Reagent Preparation) to the cells (0.5-1 mL per 10 cm tissue culture plate).
4. Place the culture plates on ice for 10-20 minutes.
5. Detach the cells from the plates by scraping with a cell scraper.
6. Transfer the lysates to appropriate size tubes and place on ice.
7. If nuclear lysis occurs, the cell lysates may become viscous and difficult to pipette. If this occurs, lysates can be passed through a 27½-gauge syringe needle 3-4 times to shear the genomic DNA.
8. Clear the lysates by centrifuging at 12,000 x g and 4°C for 10 minutes.
9. Collect the supernatant and store the sample (~1-2 mg of total protein) on ice for immediate use, or snap freeze and store at -70°C for future use.
Adherent Cells
1. Culture cells and stimulate with activator or inhibitor as desired.
2. Perform a cell count and then pellet the cells through centrifugation.
3. Aspirate the culture media and wash twice with ice-cold PBS.
4. Completely remove the final PBS wash and add ice-cold 1X Assay/Lysis Buffer (See Reagent Preparation) to the cell pellet (0.5-1 mL per 107 cells).
5. Lyse the cells by repeated pipetting.
6. Transfer the lysates to appropriate size tubes and place them on ice.
7. If nuclear lysis occurs, the cell lysates may become viscous and difficult to pipette. If this occurs, lysates can be passed through a 27½-gauge syringe needle 3-4 times to shear the genomic DNA.
8. Clear the lysates by centrifuging at 12,000 x g and 4°C for 10 minutes.
9. Collect the supernatant and store sample on ice for immediate use, or snap freeze and store at -70°C for future use.
C. In vitro GTPγS/GDP Protein for Positive and Negative controls
Note: In vivo stimulation of cells will activate approximately 10% of the available Rap1, whereas in vitro GTPγS protein loading will activate nearly 90% of Rap1.
1. Aliquot 0.5 mL of cell extract (or 1 µg of purified Rap1 protein) into two microcentrifuge tubes.
2. To each tube, add 20 µL of 0.5 M EDTA (final concentration of 20 mM).
3. Positive control: add 5 µL of 100 X GTPγS (Cat. # 30302) to the 1st tube
4. Negative control: add 5 µL of 100 X GDP (Cat. # 30304) to the 2nd tube.
5. Incubate both tubes at 30°C for 30 minutes with agitation.
6. Stop loading by placing the tubes on ice and adding 32.5 µL of 1 M MgCl2 (final concentration of 60 mM).
D. Affinity Precipitation of Activated G Protein
1. Aliquot 0.5-1 mL of cell lysates (about 1 mg of total cellular protein) to a microcentrifuge tube.
2. Adjust the volume to 1 mL with 1X Assay/Lysis Buffer (See Reagent Preparation).
3. Add 1 µL anti-Rap1-GTP antibody (Cat. # 26912).
4. Prepare the protein A/G Agarose bead slurry (Cat. # 30301) by resuspending through vertexing or titrating.
5. Quickly add 20 µL of resuspended bead slurry to above tube.
6. Incubate the tube at 4°C for 1 hour with gentle agitation.
7. Pellet the beads through centrifugation at 5,000 x g for 1 min.
8. Aspirate and discard the supernatant (making sure not to disturb or remove the bead pellet.
9. Wash the beads 3 times with 0.5 mL of 1X Assay/Lysis Buffer, centrifuging and aspirating each time.
10. After the third wash, pellet the beads through centrifugation and carefully remove all the supernatant.
11. Resuspend the bead pellet in 20 µL of 2X reducing SDS- PAGE sample buffer.
12. Boil the sample for 5 minutes.
13. Centrifuge it at 5,000 x g for 10 seconds.
E. Western Blot Analysis
1. Load 15 µL/well of pull-down supernatant to a polyacrylamide gel (17%). It is recommended to include a pre-stained MW standard (as an indicator of a successful transfer in step 3 below).
2. Perform SDS-PAGE following the manufacturer’s instructions.
3. Transfer the gel proteins to a PVDF or nitrocellulose membrane following the manufacturer’s instructions.
Note: Steps 4-11 are at room temperature with agitation
4. Following electroblotting, immerse the PVDF membrane in 100% Methanol for 15 seconds, and then allow it to dry at room temperature for 5 minutes.
Note: If Nitrocellulose is used instead of PVDF, step 4 Should be skipped.
5. Block the membrane with 5% non-fat dry milk or 3% BSA in TBST for 1 hr at room temperature with constant agitation.
6. Wash the blotted membrane three times with TBST, 5 minutes each time.
7. Incubate the membrane with Anti-Rap1 Rabbit Polyclonal Antibody (Cat. # 21156), which is freshly diluted 1: 50~500 (depending on the amount of Rap1 proteins in your sample) in 5% non-fat dry milk or 3% BSA in TBST, for 1-2 hr at room temperature with constant agitation or at 4°C overnight.
8. Wash the blotted membrane three times with TBST, 5 minutes each time.
9. Incubate the membrane with a secondary antibody (Cat. # 29002), which is freshly diluted 1: 1000 in 5% non-fat dry milk or 3% BSA in TBST, for 1 hr at room temperature with constant agitation.
10. Wash the blotted membrane three times with TBST, 5 minutes each time.
11. Use the detection method of your choice such as ECL.
同类推荐
更多
工商信息
企业名称
武汉费斯德生物科技有限公司
企业信息已认证
企业类型
有限责任公司(自然人独资)
信用代码
91420103MA7H14773E
成立日期
2022-01-19
注册资本
壹佰万圆整
经营范围
一般项目:细胞技术研发和应用;生物化工产品技术研发;工程和技术研究和试验发展;自然科学研究和试验发展;医学研究和试验发展(除人体干细胞、基因诊断与治疗技术开发和应用);技术服务、技术开发、技术咨询、技术交流、技术转让、技术推广;销售代理;实验分析仪器销售;货物进出口;技术进出口;进出口代理;化工产品销售(不含许可类化工产品)(除许可业务外,可自主依法经营法律法规非禁止或限制的项目)
联系我们
武汉费斯德生物科技有限公司
公司地址
武汉东湖新技术开发区高新大道666号武汉国家生物产业基地项目（生物创新园）B.C.D区研发楼B3-3楼301室
客服电话
Rap1 活性检测试剂盒/高校推荐厂家由武汉费斯德生物科技有限公司为您提供，货号81401，规格：30 Assays，CAS号：，如您想了解更多关于Rap1 活性检测试剂盒/高校推荐厂家价格、Rap1 活性检测试剂盒/高校推荐厂家结构式、批发、用途等信息，欢迎咨询。除供应Rap1 活性检测试剂盒/高校推荐厂家外，还可为您提供 Anti-IDH2(R172K) Mouse Monoclonal Antibody/现货抗体2023更新文献、RhoA-GTP 小鼠单抗/现货抗体《推荐》、Gα13活性检测试剂盒/2022试剂盒更新《推荐》等试剂，公司有专业的客户服务团队，是您值得信赖的合作伙伴，武汉费斯德生物客户服务电话，售前、售后均可联系。
推荐产品
供应产品
联系方式：
公司名称： 武汉费斯德生物科技有限公司
公司地址： 武汉东湖新技术开发区高新大道666号武汉国家生物产业基地项目（生物创新园）B.C.D区研发楼B3-3楼301室 联系人： 黎敏 邮编： 430074
主营产品：
友情链接：
仪器信息网APP
展位手机站