I have cloned Hemagglutinin (HA1 only) with 6xHis on its C-terminus into pet23C and expressed in E.colli (Bl21(DE3)) and then purified on Ni-NTA column, but my protein after purification had higher molecular weight. I performed Western blotting with monoclonal antibodies to His tag and antibodies binds to shifted protein in specific manner, without non-specific bands. Also i had controls before purification (lysate) and after (washes). HA1 protein in lysate had his normal weight and also binds with antibodies in specific manner. Why protein shifted to higher molecular weight?
Hi Dilyara,
Have you used fresh reducing agents? I will recommend you to use 1mM DTT. Some time inter S-S bonds can form oligomers, even when you denature your sample by boiling and in the presence of SDS loading dye.
Try to use fresh reducing agent first, if you are still seeing the shifting, send your protein sample for mass spec to analyze the molecular mass.
Dilyara Gritsenko I hope you are calculating the molecular weight of the protein along with His tag molecular weight. Usually the molecular weight of the protein along with his tag would be more than the actual protein. if its 6x his tag then you need to add around 1kDa to your actual protein molecular weight. and if the molecular weight if way too high then please check for aggregation or dimerization or attracting the dye molecules etc.
Bassam Kudhair Thank you for answer. I don't have shifting problem with HA protein in lysate, before purification. Reagents and conditions of SDS-PAGE are the same for all samples.
Beera Shankar Anand Thank you for answer. Yes, i calculated MW of my protein with His tag, and i don't have shifting problem with HA protein before purification. MW of HA in lysate is normal.
I performed purification under denaturing condition, all buffers contain 8M Urea. HA proteins were eluted with pH 5,9 and 4,5 buffers.
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