Integral plasma membrane proteins can aggregate during heating, and can be lost to the plastic of the tube, if the tube is not siliconized. Often this looks like smearing on a gel/blot. I received a protocol from a colleague who worked on a serpentine receptor. This protocol uses a sample buffer that includes 8M urea and 5% SDS (remaining components are 40 mM Tris pH6.8, 0.1 mM EDTA, 0.4 mg/ml bromphenol blue, and 1% beta-mercaptoethanol); samples are heated to 42oC for 10 or 15 minutes instead of boiling. It usually works well.

I've read that instead of boiling with SDS, one can incubate the proteins with loading buffer @70°C or even 37°C for extended time (I think it was 1 hour for 37°C), which should be sufficient.

But I personally haven't seen much difference when I tried it once.

Integral plasma membrane proteins can aggregate during heating, and can be lost to the plastic of the tube, if the tube is not siliconized. Often this looks like smearing on a gel/blot. I received a protocol from a colleague who worked on a serpentine receptor. This protocol uses a sample buffer that includes 8M urea and 5% SDS (remaining components are 40 mM Tris pH6.8, 0.1 mM EDTA, 0.4 mg/ml bromphenol blue, and 1% beta-mercaptoethanol); samples are heated to 42oC for 10 or 15 minutes instead of boiling. It usually works well.

Thanks for the responses! I'll have to try them. I am working with a handful of membrane proteins so it may come down to optimizing for each one.

I did a simple western to look at the effects of boiling and it seems that it does affect heavily glycosylated proteins more than those that aren't.

For many years in grad school I kept getting no signal on western blots of a membrane transporter protein.  We tried 3 or 4 different antibodies.   Finally,  once I became a professor I read up on it more.  All those years of failure were due to one single step in the protocol.   I used to boil the samples before SDS-PAGE.  I tried incubating at 37 degrees C and all of the sudden I got an excellent  signal for my membrane protein.   It was confirmed with an expressing cell line and an empty vector transfected cell line.   The band was strong in one and absent in the other.   That one simple step  (37 degrees for 30 min with loading buffer and beta-mercsptoethanol) gave me a consistent and strong signal every time.   I could finally stop blaming the antibodies.  So,  no boiling is my default when it comes to membrane proteins.   That's true in denaturing conditions, as was the case for the antibodies that I used in this example. 

Following your answers, for extracting a transmembrane protein from yeast, which often involves a 8M urea 65C 10 minutes heating (that also accounts for membrane lysis in this extraction protocol), is this temperature okay in the presence of high urea, or membrane proteins are still prone to urea-resistant aggregation in such elevated temperatures?

For mammalian cells I always use 2% SDS in my RIPA buffer nowadays, and I always lyse on ice with intermittent agitation via vortex. I continue this lysis until centrifugation yields NO pellet whatsoever. Sometimes it can take more than an hour. but eventually everything will dissolve, and heat should not be necessary. I would never use high heat again with a membrane protein.

I used to work with membrane transporter that was heavily glycosylated (MRP1/ABCC1) and non boiling the samples but incubating for 1h at 37C was the trick to be able to detect it!

Thank you guys for your answers, seeking a few clarifications...

Elzbieta I Stolarczyk What were the components of your lysis buffer? I work with MRP-1 too and the western blots are still so frustrating even after i switched to 37C for 1 hr.

Adam L Vanwert Your procedure was not very clear to me, '' I continue this lysis until centrifugation yields NO pellet whatsoever''. Do you mean that you resuspend pellet into the lysis solution after every centrifugation until there is no pellet?

TIA.

@Caroline, yes we had to resuspend a few times and prolong the lysis. However, in my last attempt I used the needle method. I used a 27 gauge needle and pulled the thick suspension through quickly just a few times. It gives the consistency of water after just about 5 or so times through the needle. We found that this needle method is quicker and safer (less risk of overheating sample) than sonication. The main point is that it is possible to run a sample on SDS-PAGE without discarding any portion of the cell lysate. If you discard anything (in a pellet) you will not know if your protein is in the pellet unless you test it directly.

Thank you so much Adam, will post a response after trying it out.

@Caroline Wanjiru Muriithi, did you have any luck?

Dear Adam L Vanwert ,

Thank you for your suggestions! I am working on a big GPCR, 150kd + 30kd tag. And I never able to see a band. I am wondering what is your ripa buffer recipe? I have one ripa buffer with 0.1% SDS, should i just add more SDS to 2% and lyse the cells? Thank you so much!

Hi @Xue Yang,

I don't have my recipe handy right now, but it's standard, except the 2% SDS. It gets really foamy if you pull through a 27 gauge needle, but it seems to lyse cells very quickly that way. I centrifuge at about 20,000 x g mostly to remove most of the foam. It always turns from viscous to watery with the needle method. The SDS can precipitate on storage after a few weeks (beautiful white needles) , but just warm it up in the microwave to dissolve again. Then it's a good idea to cool the buffer before adding protease inhibitors. I never have a pellet anymore because everything dissolves, and the gel still runs well (nothing stuck at the top, which used to happen to me when I boiled and had low SDS).

Good luck!

Adam L Vanwert Thank you so much Adam! I will try next week with your method!!

Adam L Vanwert It worked, Thank you so much! My sample is too viscous, and it was hard to become watery with 27g needles treatment. So I decided to go with sonication. Both needles and sonication worked for me, just the needle ones is sticky, but the bands are still there! Much appreciated!

@Xue Yang, I'm so happy it worked! It's a very frustrating thing to have something simple like that fail. It's so great to hear it works for GPCRs. Do you mind sharing what you are researching?