Membrane proteins are poorly recovered in aqueous buffers, so RIPA would be a poor choice for membrane protein solubilization. Ideally, you would want to prefractionate the tissue without denaturing/reducing agents and resuspend the membrane in a detergent free or mild lysis buffer to keep the ion channel structure (e.g. the tetramer of subunits that comprise the channel) intact as long as possible.

Prefractionation, or subcellular fractionation, can be performed via a number of ways, including sucrose gradients and/or serial ultracentrifugation, among others. If you do not have the means to run sucrose gradients, serial ultracentrifugation is simple and effective. I base my fractionation off the following paper, which is a good starting point:

http://www.ncbi.nlm.nih.gov/pubmed/15602774

A few things to consider when working with transmembrane proteins (which tend to be larger): Boiling membrane samples can cause aggregation of transmembrane proteins, resulting in messy smears at the top of the gel. After adding SDS loading buffer, try heating the sample at 50 to 70 deg C for 15 or 10 min, respectively.

If you are looking at a simpler method, CHAPS is a zwitterionic detergent said to solubilize membrane proteins quite well. However, I have not used it and can't tell you much more about it.

If you're having problems detecting membrane proteins on your nitrocellulose (NC) or PVDF, try heating the NC or PVDF in SDS posttransfer. See the following article:

http://www.sciencedirect.com/science/article/pii/S0003269708006829

Best of luck!!

Personally, I would not recommend Normal RIPA buffer for trans-membrane protein solubilisation. lysis buffer containing Nonidet™ P 40 Substitute would be the best choice. I have been doing WB of membrane proteins, tried different buffers, however I got very nice bands only with buffer containing Nonidet™ P 40. If you want to go for commercially available lysis buffer then I will strongly recommend N-PER (Thermo).

I hope this information will be useful for you. good luck

Membrane proteins are poorly recovered in aqueous buffers, so RIPA would be a poor choice for membrane protein solubilization. Ideally, you would want to prefractionate the tissue without denaturing/reducing agents and resuspend the membrane in a detergent free or mild lysis buffer to keep the ion channel structure (e.g. the tetramer of subunits that comprise the channel) intact as long as possible.

Prefractionation, or subcellular fractionation, can be performed via a number of ways, including sucrose gradients and/or serial ultracentrifugation, among others. If you do not have the means to run sucrose gradients, serial ultracentrifugation is simple and effective. I base my fractionation off the following paper, which is a good starting point:

http://www.ncbi.nlm.nih.gov/pubmed/15602774

A few things to consider when working with transmembrane proteins (which tend to be larger): Boiling membrane samples can cause aggregation of transmembrane proteins, resulting in messy smears at the top of the gel. After adding SDS loading buffer, try heating the sample at 50 to 70 deg C for 15 or 10 min, respectively.

If you are looking at a simpler method, CHAPS is a zwitterionic detergent said to solubilize membrane proteins quite well. However, I have not used it and can't tell you much more about it.

If you're having problems detecting membrane proteins on your nitrocellulose (NC) or PVDF, try heating the NC or PVDF in SDS posttransfer. See the following article:

http://www.sciencedirect.com/science/article/pii/S0003269708006829

Best of luck!!

I can only tell you what we use for mouse brain.

Mix tissue in PBS with polytron, centrifuge 10min 3000g (4°C), then homogenize the pellet with dounce homogenizer in homogen. buffer: Tris-HCl 50mM pH 7.4, EDTA 0.5mM, CHAPS 1.3%, protease inhibitor cocktail. Incubate on a wheel 2-4h at 4°C, then centrifuge 30min 30 000g (4°C) and use supernatant.

CHAPS is good for transmembrane proteins, I use it for 11-transmembrane domains transporter. You need to incubate a few hours on a wheel at 4°C to have an optimal lysis, though. You still get a pellet with unsolubilized material (DNA and other stuff, I guess), but it's one of the most efficient detergent for transmembrane proteins.

For more personnalized detergents, you can try also these commercial ones:

http://www.logopharm.com/products-and-services/complexiolyte-buffers

they sell a range of detergents, from low to high stringency, that you can try to see which is best for your favorite protein.

And I agree that heating is a bad idea for transmemb prot. For example at 95°C Glut2 glucose transporter aggregates and can not even enter a polyacrylamide gel. Usually I use 70°C if I need to heat (to elute after an IP, for ex.).

Try doing some tests with/without heating, and with different buffers before doing the real exp. Good luck!

RIPA buffer would be fine. good luck!

Ke. Please explain why RIPA is sufficient.

Hi Grant, I mostly agree with Grant's and Pascal's answers. RIPA only is not a good idea.

May I add that you can also consider doing subcellular fractionation, if you are interested in a synaptic protein, After homogenization you can isolate synaptosomes/synaptic membranes. It always helps to know where the protein is located. Please be aware that this can lead to quite some loss in sample, so make sure you have enough material.

Please refer to https://www.sciencemag.org/content/327/5972/1518.abstract for fractionation protocols.

I would not go for RIPA extraction alone. Instead i would go for a membrane extraction protocol like described in "Li et al. 2004, Proteomics analysis of rat brain postsynaptic density. Implications of the diverse protein functional groups for the integration of synaptic physiology.", so you can enrich your membranes containing ion chanels.

If you want to isolate membrane-associated protein you need to centrifuge at 100,000xg. If you just want to homogenize your sample you just can do it but you cannnot state that you are detecting membrane-associated channel. There are several protocols that allow to separate membrane from cytosol. We used a differential centrifugation protocol in the past. Now we used a Termo-Fisher kit that work really well (Please see ref. J Psychopharmacol. 2012 Oct;26(10):1333-47.) Good luck!!