Hi all,
I have a protein supposed to be around 20 kDa, but somehow on SDS-PAGE it runs at 40 kDa and previous literature also showed the same behavior and suggested a dimer complex. I am little bit confused, since it is a denaturing gel (4-20% tricine gel). I have sent the protein solution for mass-spec as well as the protein band. I am wondering if mass-spec can tell the protein could form a covalent bonded dimer? If so, how do I obtain that information or do I need any special mass-spec?
Intact protein liquid chromatography-mass spectrometry performed under denaturing conditions will show whether the complex is covalent. If the covalent bond is a disulfide bond, SDS-PAGE under reducing conditions should show only the monomer.
Adam B Shapiro Hi Adam, thank you for your answer. Unfortunately, the protein does not contain any cysteine, thus it cannot form disulfide bond. I also added reducing agent during SDS-PAGE gel. The 40 kDa was confirmed by Western, that's why I am super confused. I am wondering is there any other possible covalent bonds happening besides disulfide bond?
Sebastian Schmitt
Hi Sebastian,Thank you for your answer. The protein is an extracellular protein from extracted from cell-free media. I did not add any tag and the Western was done on whole cell lysate (published result). I agree there might be many troubles with it. But I have a medium resolution structure (~5 A) which suggested that this protein cannot be a dimer. That's why it confused me. But I agree it might be due to post-translational modification.
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