Hi

Membrane proteins are hydrophobic in their core so you need a lysis buffer that contains urea to disrupt the membrane and solubilize them.

http://cshprotocols.cshlp.org/content/2006/1/pdb.rec9992.full?text_only=true

remember that boiling samples at 95C is not recommended because it introduces protein aggregation in such proteins. Instead you can incubate them at 40C for 30 min in 4x Laemmli Sample Buffer that that is accompanied with 8M urea.

Best

I have no experience with your stuff however Triton-X114 might be helpful in your case.

If you add proper amount of Triton-X114 to your extraction buffer, it will separate above 22°C into two phases so that you will have in dense phase mainly membrane proteins which are not soluble in water while less dense phase will include less detergent and mostly water-soluble proteins

Hi Abhishek, I have some experience in preparing cell extracts for membrane protein detection for WB.

1. Wash the cell layer with cold PBS (3x10 ml should do it) Drain the plate of excess PBS.

2. Add 1 ml of SDS sample buffer (if you have a 150mm2 plate)

3. Scrape the cells on the plate onto one side.

4. Use a syringe with a small bore needle to suck up and expel the lysate until the DNA is sheared. (if the cell lysate is very thick, you can start off with a larger bore needle and then switch to small bore needles (tuberculin syringe needles are ideal).

5. You know when the lysates are ready when you can use regular pipette tips easily without any sticky stuff clinging to them or clogging.

@Steingrimur Stefansson : Thank you for your response. I have tried with SDS sample buffer but i have not use syringe afterwords. I have loaded the sample by heating it to 95 C and followed by centrifugation at 13000 rpm for 10 min. soup as well as pellet I loaded but still i got nothing.

Farjam Goudarzi : Thank you for your response. I have used RIPA buffer

10mM tris cl

1mM EDTA

0.5 mM EGTA

1% Tritin X-100

0.1% sodium deoycholate

0.1% SDS

140mM NACL

which is very harsh buffer which is used for membrane proteins. I have also Incubated cell lyasate at 37 C for 15 min to check whether this works or not. But unfortunately it dint work. I will try with 8M urea.If this work i will let you know.

Erman Kocak : Thank you for your response. I would definitely try this out but can you suggest me tentative concentration or percentage i will be adding in lysis buffer?. Thank you

I would suggest 2% working concentration due to its cloud point at 1%

you should work at low temperature (4 °C?) then leave it at 22 °C until you see a phase separation.

Erman Kocak Thank you

Hi Abhishek, if you shear the DNA in SDS sample buffer with a syringe needle you can load it directly onto a gel without centrifugation.

Heating the sample might destroy the Ab epitope in WB.

Steingrimur Stefansson Erman Kocak Farjam Goudarzi

Thank you all for your input. My problem was solved later. I had to use higher anti-RAGE antibody concentration 1:1000 for RAGE(receptor of AGE) & even I have to use the same concentration of Anti-FLAG antibody to get that band. Normally for FLAG-Ab 1:7000 works well. But i am happy that i can get my band. I guess the protein concentration may be less or I should check protein concentration in membrane fraction?.Thank you.