Can anyone help me - I mutated protein, (point Ala mutation) so the products should be the same molecular weight. One of them is mutated correctly (1st mutant) but the other one didn't gave the expected product. After PCR I did the Miniprep and sequenced the product - everything is okay, mutation is present. I tested different strains for expression and the only product I managed to obtain is this one (2nd mutant) with the higher molecular weight.
Is it possible that during mutagenesis, some spontaneous mutation occured and the reading frame was shifted?
Hi, Malwina!
Nonspecific mutations have a rather high probability during site-directed mutagenesis but you can easily find out whether this is the case with your 2nd mutant by sequencing the entire ORF of your mutated gene. A frameshift, insertion or duplication would explain the migration that you observed. However, it is also possible that there are no spontaneous mutations, but the point mutation of your 2nd mutant changes the hidrophobicity or the electrostatic properties of your protein in a way that slightly alters its migration. You can read more about this in this paper: "Abnormal SDS-PAGE migration of cytosolic proteins can identify domains and mechanisms that control surfactant binding" by Shi et al., 2012.
I would recommend first to sequence the ORF of your gene and than if everything is ok and you have no unspecific mutations, to make a western blot with an antibody against your protein of interest.
Good luck!
Hi, you should first double check that there are not further mutations in your construct. Also, that you made the construct correctly and in frame. Once all that is double checked, is posible that a a single point mutation destabilize a protein, so that you find less/undetectable protein.
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