| | Re: SDS Boiling of Samples Any Alternative Methods?
As far as I know, boiling was introduced to combat proteolysis. The idea being that as SDS unfolds the proteins in the sample any protease there will have a 'field day' for the time spent at near optimum temperature with an unfolded substrate to act on. It is essential, therefore, to get the sample to a temperature where any proteases present are instantly inactivated. Probably, there is no need to go to 100 degrees C, but 40 degrees C for 30 min seems crazy to me, unless the risk of proteolysis can be completely excluded.
For this reason also, you should always add HOT sample buffer to the sample, especially with crude extracts.
There are a few other points. Do you centrifuge you samples prior to loading? This usually gets rid of precipitation.
Do you have a stacker gel? It is not uncommon for high molecular weight proteins not to enter the gel. But if you have a stacker (say 4 percent or something like that) this is an unlikely explanation.
Is there a lot of nucleic acid present? Are your samples 'gloopy'. If this is the case, you may need to sonicate.
I suppose the answer is there is no need to boil, but beware of proteolysis