Lab Techniques

Hi all, SIP/MP is finally over!!

Today I'm going to talk about the extraction of cell membrane proteins. Jiaxin had described how we extracted secretory proteins using TCA/Acetone precipitation method. For the extraction of cell membrane proteins, we are using ReadyPrepTM Protein Extraction Kit (Membrane I) from BIORAD.

Picture is taken from http://www.biocompare.com/images/spotlight/spot_isc205_biorad.jpg

Principle:

The membrane proteins are extracted based on temperature-dependent phase partitioning. The sample is first mixed and homogenized in the extraction buffers (buffer M1 and M2) provided in the kit. After a brief incubation at 37oC, the sample is centrifuged at 28oC to produce 2 phases. The top layer (hydrophilic) will contain cytoplasmic proteins, bottom phase (hydrophobic) will contain the membrane proteins and an insoluble pellet would contain the complex membrane proteins that are not solubilized by the extraction buffers.

The components of the extraction buffers are not revealed by the manufacturer and thus the exact mechanism is unknown.

Procedures:

The first 3 days are the same (Refer to Jiaxin's last post).

On the 4th day (Extraction of proteins using the kit)
- After the supernatant is used for extraction of secretory proteins, the cell pellet will be used for labelling with Cy3. Extraction of the proteins will only start after the labelling is done.

1. Centrifuge the tube at 5000xg for 20 min at 4oC (to pellet the cells)
2. Transfer the supernatant to a new 1.5mL eppendorf tube and store
3. Add an appropriate volume of buffer M1(0.5mL to 0.05mL of cell pellet) to the cell pellet and resuspend
4. Add an appropriate volume of protease inhibitor(1uL to 100uL of buffer M1) to the resuspended sample
5. The suspension was splited equally into 5 tubes (if not subsequently there will be not enough space to add buffer M2)
6. Sonicate the suspension for 35 sec (to lyse the cells)
7. Chill the suspension on ice for 1 min (sonication would produce heat)
8. Repeat steps 6 and 7 for 6 times
9. Add an appropriate volume of buffer M2(equal volume to buffer M1) to the suspension and mix well
10. Vortex the suspension for 1 min
11. Chill the suspension on ice for 1 min
12. Repeat steps 10 and 11 for 7 times
13. Chill the suspension on ice for 10 min
14. Transfer the suspension to 37oC water bath for 30 min (Invert tubes thrice for 30 sec to mix the suspension)
15. Centrifuge the tubes at 16,000xg for 5 min at 28oC
16. Transfer the top layer(containing hydrophilic proteins) to a new 2.2mL eppendorf tube and store
17. Add an appropriate volume of buffer M2(equal amount as step 9) to each of the bottom phase and mix well
18. Repeat steps 10 to 16
19. Transfer the bottom phase(containing the hydrophobic proteins) to a new 2.2mL eppendorf tube and store

The bottom phase which contains the hydrophobic proteins is our interest as most membrane proteins are hydrophobic.

That is all! Thanks=)

Chua Ming Boon
0503197F
TG01