A colleague prepared Western blot from a protein which should normally appear only as one band on the Western blot Membrane. The concentration of the protein is low, BCA test negative.
What she saw is many smears, no clear band and maybe it looks like two different bands.
Does anybody know this phenomenon? See the picture for details.
If you see like a smear it is possible that your protein is degraded.
Hello Lahiri,
thanks for your comment. I just asked my colleague and she says that the antibody is against the HIS-tagged Protein of interest. So it should be specific.
If you see like a smear it is possible that your protein is degraded.
was that protein purified? If that protein was purified, it means that protein got degraded , or may be it happens when proteins are not purified and so many proteins appears on western blot membrane...
The protein was purified in three steps. First step is ion exchanger (DAE), second gelfiltration and third is affinity chromatography. After all steps the Westerblot shows the same picture. In this case the picture was taken after step one.
Did you know the reason why the protein can be degraded?
Degredation could happen for a number of reasons: 1. perhaps there is ligand that stabilizes the protein and may be needed along with the purification. 2. Purification of proteins utilizes a number of steps and buffer conditions that may not be ideal for the protein of interest. 3. Interactions with purifications columns may partially unfold a protein. The degredation of the protein is a common problem when using multi step purification schemes.
Is the higher band or lower band the predicted MW for the protein. If it is the higher band, then it is likely degradation. If it is the lower band, is this a glycoprotein that may be glycosylated? It could also be a dimer if the gel was not run under denaturing and reducing conditions, but again only if the higher band is larger than expected.
Thanks again for the answers.
@Aaron: Thanks for telling possibilties for the degredation
@James: Yes, it is the higher band which shows the protein of interest
Anybody ideas what we can do to reduce the degradation?
you should use fresh buffers to avoid protein degradation. even during purification also, you should use fresh buffers. Temperature maintenance is most important step, protein should be in ice even during loading on SDS-PAGE. we should avoid thawing protein by hands!!
Since now we assume that is more likely to be a degradation than Ab unspecificity, I think your friend should find in which step of the process occurs the degradation. Since the degraded pattern is evident after the first chromatographic step, the protein may be being degraded in a previous step. Did she use any protease inhibitor in the lysis buffer? if not she may add 0.5-1 mM PMSF, that would help prevent proteolysis
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