I used both like heating sample with sample buffer at 95 to 99'C for 5 to 7 min or kept for 5 min on boiling water. The purpose of this is the same but which is the correct method?
You have to be careful when using heat to denature proteins because they can aggregate. Proteins with trans-membrane domains or multi-pass membrane proteins tend to aggregate when boiled. In this case heating at 70°C for 5-10 minutes is preferable otherwise 90°C for 5 min should be sufficient. Overall it all depends on the protein's structure.
I myself use the first one and it works but for 10 minutes. I think the second one is also right, but might be a traditional one.
You have to be careful when using heat to denature proteins because they can aggregate. Proteins with trans-membrane domains or multi-pass membrane proteins tend to aggregate when boiled. In this case heating at 70°C for 5-10 minutes is preferable otherwise 90°C for 5 min should be sufficient. Overall it all depends on the protein's structure.
Denaturing at 95C on a heating block for 3 minutes prior to standard SDS-PAGE is sufficient in most cases. However, if your protein of interest contains TM domains, I'd recommend not heating at all, but preparing your lysate in lysis buffer containing protease inhibitors. Bear in mind though, that if you don't boil your samples they may be very viscous, which can cause problems when loading - I routinely vortex my samples prior to loading, which reduces the viscosity sufficiently for my purposes.
Denaturing samples at 95 C for 10 min on a heating block is better. And it works for most of the proteins. Every time, I spin the samples for few seconds and load them.
Are you boiling protein samples in Laemmli buffer to prepare for SDS-PAGE? The attached article may help you. In particular, it is important to heat protein samples immediately after addition of Laemmli buffer, to prevent degradation of denatured proteins by some proteases which can be (partially) resistant to SDS-denaturation. Also, prolonged heating at high temperatures can lead to acidic proteolysis. Sometimes you have to try a range of different temperatures (eg room temperature, 50/70/95C) and times (1-10 minutes) to see what works best for your protein of interest.
I always used heat block but right now I wanted to discuss the roll of Boil and Heat with reference to protein nature. Thanks to all for participating in the discussion
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