I cloned my gene of interest in pET28a vector, my protein molecular weight is 24 kDa but its coming at 27 kDa, why is there such shifting in SDS PAGE?
Hi Ajeet
Have yo considered the sequence in the vector pET28a prior to the 'ATG' of your gene....those sequences inclusing theHIS tag will also contribute to the size of your protein....
bye
Hi Ajeet
Have yo considered the sequence in the vector pET28a prior to the 'ATG' of your gene....those sequences inclusing theHIS tag will also contribute to the size of your protein....
bye
Hello Ajay,
I have checked it,that sequence contribute around 1kDa but still shifting is 2-3 kDa is more,what is the reason?
If it is Histidine tagged (6X), these proteins usually have retarded mobility on PAGE. If it is recognized by your protein specific antibody in western blot, it should be fine.
This small shift is in the range of accuracy of SDS-PAGE. If you are using prestained MW marker, remember the size of the band is lot and age dependent. Don't worry, I would go on with your prep!
I think it is probably fine. Sometimes proteins do migrate bigger than their molecular weight. This happens when the protein is highly charged (like RNA binding proteins for example). Of course, if you are purifying it for biophysical characterisation I would sent it off for mass spec.
Hi! Some proteins will migrate anomalously on SDS-PAGE because of the altered binding ratio of SDS molecules to the unfolded peptide. There are some structural motifs that would enhance this "weird" behavior. The best example is the famous p53 that is supposed to be 44kD aprox and runs as if it was 53... Anyway you shouldn't worry and do a mass spec if you are not completely sure, as Luigi says.
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