I have always kept raw samples on ice until the addition of SDS sample buffer. If the samples were frozen in SDS sample buffer, I have never thawed on ice. I keep them at room temperature once they have SDS sample buffer added.

Carly Wiersma Thanks for your insightful comment! Is there any standard protocol available for this, like from Biorad or any other authentic source?

Md Rafi Anwar Not that I know of or could find. This article explains the science behind the buffer if that helps: https://advansta.com/ask-advansta-why-do-we-heat-protein-samples-before-loading/

I can tell you that SDS is a detergent that we add because it causes the proteins to become linear by denaturing them which . It also coats the proteins in a negative charge so that the protein will run the correct direction on the gel (negative to positive). Since SDS disrupts protein structures, that includes proteases that may degrade your protein, so that is why it is okay to keep them at room temperature in SDS sample buffer (temporarily) before you run your gel. I wouldn't try leaving your samples overnight or anything in SDS sample buffer, but a couple hours is okay generally. I store my samples in SDS sample buffer at -80.

Carly Wiersma Thank you very much indeed for your comprehensive explanation.

It is always better to cool them down. However, if you are not able to cool samples befor adding to the gel, it should also work.

Andreas Eisenreich Thanks for your answer

Your sample is stable enough to keep a short time at room temperature AFTER adding sample buffer AND heat denaturation. Therefore, after thawing from -80 or -20, you can just warm up @ 40 C to redissolve the SDS, then vortex and spin down. Then you can leave on the bench for the time required to load. Be careful to put the sample on ice at this stage, as SDS precipitates with low temperatures