I ran a SDS-PAGE of my sample ovalbumin in histidine buffer in 2x Laemmli buffer without heating and it got nice visible bands. However, when I heat the sample at 77C in 2x laemmli buffer for 5 min and ran SDS-PAGE again, the bands become so light that I can barely see them.
Either case I use commasie blue to stain 4 hours. With heating, there is no blue build up on the top of the gel which indicates the protein didn't aggregate during heating and went through the gel. So there should not be any clogging in the gel I think. And, 2x Laemmli buffer shall provide me sufficient SDS to prevent aggregation.
Hello, partial thermal denaturation of ovalbumin occurs at 78 to 86 °C. The protein adopts a more stable form, termed S-ovalbumin to denote its increased stability. The 77 °C that you mention can cause partial denaturation of ovalbumin which can be reason of problem that yoy mentioned.
It should be because of the amount of CNBr used, there should be a precised amount based on the quantity of the sample u are working with. Check your journal guide very well or rescale values
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