Hi. I prepared my protein samples. After boiling them I realized that i added less loading buffer to a couple of samples than i had calculated. But now its already boiled. Can i add more loading buffer to it and boil it again? Or will that denature the proteins completely?
I assume you are talking about SDS-PAGE samples?
Depends on the volume missing; if it is just a few percent of the total volume, I would just add the buffer, mix and not boil them again.
The important point is that the proteins bind sufficient SDS .from the buffer. The proteins are denatured during the heating process and SDS can bind to all parts of the denatured protein. If you boiled a larger amount of protein the first time in a very tiny volume, and you suspect they were not exposed to a sufficient amount of SDS, I would boil again.
SDS-PAGE works, because practically all proteins bind on average 1.4g SDS / g protein. Therefore, you have a charge that is a function of the mass. If you have too little SDS for the protein, then they will usually run slower in the gel.
Generally, I'd avoid boiling at all, 15' at 50 °C works better especially for large proteins (> 100 kDa or so).
Hi If you are analyzing your protein using SDS-PAGE you can add more loading buffer to it and without boil it again.
You add loading buffer and boil your samples to denature all proteins.
So you can add more buffer and re-boil it.
It is good to re-boil it at 95C, since some of the proteins are thermally stable.
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