I made several point mutations on a protein, and found that only one particular Ser to Ala mutation leads to significant change in mobility of protein on SDS-PAGE (WB is attached). The drop in size is too large to suspect that is due to phosphorylation. Also when the same Ser is mutated to Asp there is slight change in mobility as well. Mutations of any other Ser did not change the mobility of protein.
Any explanations are welcome :)
Since the mutated polypeptide is significantly shorter, it represents most likely a truncated form of the protein. This may occur if the mutation destabilizes it and makes it more susceptible to proteolysis
Agree with Pierre.. Try putting the mutated amino acid sequence in tools such as peptide cutter on expasy to find out which sequence is like to be cleave by a protease.
If needed add protease inhibitors!
I presume that these proteins are expressed in E. coli, in that case even one amino acid change can vary the protein confirmation and the ability of SDS binding that often leads to change in the mobility of the protein. Also there is a possibility of varied stability and susceptibility to proteases as Pierre mentioned.
If the proteins are expressed in yeast, then the difference in glycosylation may be one of the reason.
Best
Sunagar Raju, thanks for the answer, the protein is expressed in 293T cells, it is always extracted in the presence of protease inhibitors. From my experience glycosylation leads to smeary bands, and these were not smeary.
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