Have you sequenced your entire gene after mutagenesis? Have you run the mutant alongside the wild-type on a gel incase your molecular weight marker is not accurate? Could you have mutated a post-translational modification site that has altered the mobility?

Thanks Amanda

I run my mutant insert along with wild type that it was at correct position and I think u r right I should do sequencing so now I am doing sequencing of entire gene

Running proteins on a gel for Mw can be misleading if there are post-translational modifications or mis-folding, which could show a band at the incorrect Mw position. If you have a suitable SEC method, measuring the Mw via a first principle technique such as multi-angle static light scattering detector (DAWN HELEOS from Wyatt Technology) will provide a better Mw measurement without the assumption that your protein of interest has the same hydrodynamic properties and behavior as that of your Mw ladder.

Thanks William...

But unfortunately I didn't have that kind of techniques for Mw determination.....

and more thing William I am bacterial host system for expression so the chance of post translational modification is not possible....

Does it have a tag? and with the tag does it make it a 62kDa protein? If so then some tags can be cleaved (autocleaved) leaving the recombinant protein in it's original size .

And yes I agree with everyone else. You should sequence the gene maybe you mutated the codon into a stop codon also.

Thanks Cris Martinez

I Did SDM (amino acid replacement with other amino acid) not with stop codon and I am using GST purification technique for protein purification....my desire protein around 37 KD and GST tag is a dimer of 26 KD so my protein should be around 63kd but its coming around 52 kd.

yes sent my sample for sequencing to check the complete sequence.

possibility of degradation is also there. You never know how your protein behaves after inducing mutation.

Did you try to cleave GST tag of the 52 kDa protein and verify if it is your protein?

Thanks to all.....

yes I sent my Plasmid for sequencing and I am trying to cleave GST Tag now....I want to ask one more thing is this possible Dimer of GST tag (26kd) can change or convert into monomer (13KD) ???

monomeric GST is 26kDa and dimer is double of that.. But, if you see a prominent band at around 13kDa, it could possibly be the degradation product of your 62kDa protein giving corresponding bands at around 52 and at 10-13kDa.

Thanks Tahir

I am trying the same checking for 13 KD band (monomer of GST). in coomassie staining I didn't find it now I will try for silver staining.