Plasma Membrane Protein Extraction Kit

Plasma Membrane Protein Extraction Kit

For research use only

Plasma Membrane Protein Extraction Kit

(Catalog #K268-50; 50 preparations; Store at –20oC)

I. Introduction:

The Membrane Protein Extraction Kit provides optimized buffers and reagents for effective

extraction of membrane proteins from mammalian tissues and cells. Unlike other available

procedures that can only extract the total cellular membrane proteins (combinations of

plasma and organelle membrane proteins), BioVision’s kit was designed to not only extract

the total cellular membrane proteins, but also purify the plasma membrane proteins

specifically. The procedure offers consistent yield and high purity (over 90%). Membrane

proteins prepared using the kit can be utilized in a variety of applications, such as Western

blotting, 2-D gels, and enzyme analyses, etc. The entire procedure takes less than 1 hour.

II. Kit Contents:

III. Membrane Protein Extraction Protocol:

A. General Consideration and Reagent Preparation:

? Read the entire protocol before beginning the procedure. Be sure to keep all buffers and

reagents on ice at all times during the experiment.

? Reconstitute Protease Inhibitor Cocktail by adding 110 μl of DMSO, mix well.

? Before use, aliquot enough Homogenize Buffer, add 1/500 volume of the reconstituted

Protease Inhibitor Cocktail (e.g., Add 10 μl to 5 ml buffer) to make the Homogenize

Buffer Mix. (Note: Some precipitation may occur after adding the Protease Inhibitor

Cocktail. You may continue using the buffer or simply remove the precipitates by

centrifugation).

? The following protocol is described for extraction of ~5-10 x 108 cells. If more cells are

used, scale up the volume proportionally.

B. Extraction of Total Cellular Membrane Proteins:

1. Collect cells (5-10 x 108) by centrifugation at 600 x g for 5 minutes at 4oC.

For adherent cells, scrape cells in PBS and then spin down (3000 rpm for 5 minutes) to

pellet cells.

2. Wash cells once with 1 ml of ice cold PBS.

3. Resuspend cells in 1 ml of the Homogenize Buffer Mix in an ice-cold Dounce homogenizer

(Cat.# 1998-1). Homogenize cells on ice for 30-50 times.

For tissue samples, homogenize tissues in 2-3 volume of the 1X Homogenize Buffer, until it is

completed lysed (30-50 times).

Note: Efficient homogenization may depend on the cell type. To check the efficiency of the

homogenization, pipette 2-3 μl of the homogenized suspension onto a cover slip and observe

under a microscope. A shiny ring around the nuclei indicates that cells are still intact. If 70-80

percent of the nuclei do not have the shiny ring, proceed to the next step. Otherwise, perform

10-30 additional passes.

4. Transfer the homogenate to a 1.5 ml microcentrifuge tube. Centrifuge in 700 g for 10 minutes

at 4oC. Collect supernatant and discard the pellet.

5. Transfer the supernatant to a new vial and centrifuge at 10,000g for 30 min at 4oC.

6. Collect supernatant (This is Cytosol Fraction). The pellet is the total cellular membrane

protein (containing proteins from both plasma membrane and cellular organelle membrane).

Note: You may stop here if you only need the total cellular membrane proteins. If you would

like to further isolate the plasma membrane proteins specifically, continue with the following

steps.

C. Purification of Plasma Membrane Proteins:

7. Resuspend the total membrane proteins pellet in 200 μl of the Upper Phase Solution. Add

200 μl of the Lower Phase Solution. Mix well and incubate on ice for 5 minutes (Mark the tube

as A).

8. Prepare a fresh phase tube without samples. Adding 200 μl of Upper Phase Solution and 200

μl of Lower Phase Solution (Mark the tube as B).

9. Centrifuge both A & B tubes in a microcentrifuge at 3500 rpm (1000 x g) for 5 minutes.

10. Carefully transfer the upper phase from tube A to a new tube (tube C), keep on ice.

11. To maximize the yield, extract the tube A lower phase again by adding 100 μl of the Upper

Phase Solution from tube B. Mix well and centrifuge at 3500 rpm (1000 g) for 5 minutes.

12. Carefully collect the upper phase. Combine with the upper phase from Step 10 (tube C).

Extract the combined upper phase by adding 100 μl of the Lower Phase Solution from tube B,

Mix well and centrifuge at 3500 rpm (1000 g) for 5 minutes.

13. Carefully collect the upper phase. Dilute the upper phase in 5 volume of water. Keep on ice

for 5 minutes.

14. Spin at top speed at a microcentrifuge tube for 10 minutes at 4oC. Remove the supernatant.

The pellet is the plasma membrane protein.

15. Store the plasma membrane proteins at –70oC for further studies. The membrane fraction

can be dissolved in 0.5% Triton X-100 in PBS or other buffers before use.

Generally 1-100 μg plasma membrane proteins can be obtained.

FOR RESEARCH USE ONLY! Not to be used in humans