We found a band at the expected molecular weight and another 1-2 kD lower with almost the same intensity.
We are using an anti-DYK antibody, and our expression plasmid is pcDNA3.1+. The DYk flag is in the N-terminal of our gen.
We have tried different protocols,for harvesting and resuspend the cells using protease inhibitors, reducing agents in same cases(maybe there is a disulfide bond in our protein) and any other solution we have thought about but we still have the problem.
Maybe is due to some post-transductional modification but previous works, with the same protein and host cells have not found this problem.
We have also read the answers to a similar problem in https://www.researchgate.net/post/Double_band_observed_on_Western_Blot_of_expressed_FLAG-tagged_protein/1 and tried almost all the suggestions.
The company where we have purchased the plasmid tell us about it could be maybe related to contaminations in our plasmid when performing Maxiprep EF, but we are not sure if that could explain our problem.
We really appreciate any suggestions.
Have you thought about truncation of the protein, either through degradation or as a modification? If the bands are of similar intensity it probably is truncation.
I agree with Kumar above. Likely a truncation due to post-translational processing of the recombinant protein. One way to demonstrate this would be to immunoprecipitate the protein using your anti-DYK antibody, run an SDS-PAGE, excise the two bands of interest, and send both bands off for mass spec. analysis.
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