Dear Manasi,

As per my experience RIPA buffer can sufficiently extract membrane proteins.

Please find the attachment for RIPA buffer composition and protein extraction protocol. 

Hey Sagar, 

Thanks for your answer. I know the contents of RIPA and protein extraction. But since it is not working for my cells I was trying to assess the alternative methods if any.

Did you do reducing or non-reducing conditions for SDS-PAGE when you extracted membrane proteins?

Dear Manasi,

We use all reducing conditions for Resolving membrane proteins. We sonicate sample using two 10 second pulses (30 seconds in between pulses) using a probe sonicator on ice in RIPA buffer.

As you mentioned, you did not find any band on WB. The plausible reason is the membrane proteins are highly hydrophobic so they cant transfer easily from stacking to resolving gel. Please do not cut stacking gel during protein transfer on PVDF.

Thanks

Hi Manasi, is the antibody you use for probing the western a mono- or polyclonal?

If it is a monoclonal Ab, then it could be against a conformation specific epitope. Do you know if the mAb was made against a peptide or non-denatured protein?

Also, do you have a positive control for your westerns?

1. How do you know your protein really localize on membrane?

2. Sometimes the protein will express on cell membrane with different time courses (depend on your treatment), so perhaps you have to try diff. conditions.

3. Maybe, you could try immunofluorescence assay to make sure the localization of your protein.

Good luck~

You want to use the gentlest conditions you can.

TX100 or NP40 (1%) work well for many membrane proteins and are non-denaturing.

RIPA will pretty much dissolve everything, including nuclei, so be prepared to deal with released DNA.

One tip is not to overheat your samples in sample buffer before running the gel,  Many membrane proteins aggregate when heated. 30 min at room temp is more than sufficient.  good luck

@steingrimur  it was a polyclonal Ab and yes I did have a positive control for my westerns, although it did not work when proteins were extracted with RIPA

@Neil thanks I did try using TX100 (1%) and after a few manipulations found out that for my samples heating for 5 mins at 90C worked best ! 

Thanks everyone! :)

RIPA always causes altered protein profile and endogenous baseline due to loss of proteins non-proportionally. Strong lysis buffer should be used for complete lysis. The stickiness can be removed by using a spin column with a special filter. Sample can be ready in just a minute. Try to search Minute Protein extraction kit on Google.