Dear Igor,

I think you have a good general plan for extracting protein from the gel slices. In my experience, you can recover a very good amount of protein by diffusion. In my case, I normally let proteins elute after smashing the gel slice with a Teflon pestle and 2-4 h of diffusion in the elution buffer (normally I shake these samples at 500 RPM in a cold room, 4°C). After spinning down the gel pieces (10,000 x g), I recover the supernatant and concentrate the protein with Amicons. I’m sure that with this method I can get a yield above 70%. Since all these procedures are done at cold temperatures, the proteins remain native and functional.

If you really want to keep the proteins in their native states after diffusion, you might use this protocol instead of freezing the slices and disrupting them with a mortar. However, if your follow-up procedures are basically SDS-PAGE and WB, the native state should not be a concern for you. You can add protease inhibitors (SIGMA-FAST), 1 mM EDTA and/or 2-5 mM 6-aminohexanoic acid to keep your proteins safe throughout diffusion. For the shaking step, you can even do it overnight to achieve the highest diffusion; 2-4 h are usually enough in most cases though. You might remove the gel pieces before the acetone step.

The precipitation step with acetone looks fine, I have also read protocols that you can do this overnight. Nevertheless, I think this step is only critical if you want to remove detergents and other contaminants, potential renaturation, etc. In principle once you recover the proteins by diffusion you can just concentrate your samples and proceed for the SDS-PAGE and WB.

One last comment, you might consider doing 2D-SDS-PAGE of your gel slices and directly analyze your samples. You can even transfer the proteins from the BN-gel onto a PVDF/nitrocellulose membrane for immunodetection. Thus, you can save time and steps. Hope these comments help you! My inbox is always open if you have further questions.

Best wishes,

Alfredo

Alfredo Cabrera-Orefice, thanks a lot!

I've already done 2D BN/SDS-PAGE, and 2D BN/SDS-PAGE followed by WB. The problem is that this way I cannot use densitometry after immunoblotting - there is too much of noise and in addition Image Lab software that I use cannot properly analyse spots - only bands.

Also I've tried to WB proteins after the BN-PAGE (but using nitrocellulose instead of PVDF), but the quality of protein transfer was very poor and the membrane was stained with coomassie leftovers, so I decided to try another ways.

Alfredo Cabrera-Orefice, I need to mention another problem, which I ran into earlier during 2D BN/SDS-PAGE followed by WB - there were a significant amount of SDS-induced subcomplexes that makes measurements even more difficult. I've tried to increase the gel slices incubation time in SDS + 2-ME buffer, but it only leads to increased diffusion of BN-bands. From the other side - there are no subcomplexes after 1D SDS-PAGE.

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