Theoretically, my protein is unstable. till DNA level everything is quite fine, but I think I am losing it during heating with bromophenol for SDS-PAGE. Any suggestion?
First of all do you purify your protein or do you load a cell lysate?
I think you lose your protein earlier. It doesn't make sense to me, that you lose any protein in the boiling step.
Or do you think your protein literally falls apart?
Cheers
I think other readers will be wanting more information in order to help. There might be questions regarding why you think this protein is "theoretically" unstable, what do you mean by "losing it" in sample preparation, what type of protein and expression system do you have, can you probe the cells with any protein specific label, can you assay for transcription and transfection/transformation, etc. In other words are you asking people if there is an expression issue?
Hi there,
If you think boiling is triggering your protein degradation, boiling is actually not compulsory for SDS/PAGE sample preparation as its role is to accelerate protein unfolding which is also promoted by SDS on its own but the kinetics is longer. You may use sample buffer also containing 8M urea to help the denaturatioon at room temperature. But be aware, if using urea, all the samples loaded onto gel have to contain urea in order to get a nice migration pattern.
Just out of my own curiosity. Is it actually possible that a protein falls apart while boiling?
It is unlikely that your protein is actually degraded upon heating in SDS-PAAG buffer. However, certan proteins, particularly transmembrane ones, can multimerize upon heating in SDS buffer. Depending upon the degree of this aggregation they may form a ladder in the gel or simply fail to enter the gel from the loading well. Possible remedy for this is described above by Dominique.
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