I am trying to purify His-tagged protein (by Ni-NTA resin) after over-expression in BL21 cells. When i run SDS-PAGE of un-induced, induced, Lysate, supernatant, pellet and purified protein samples, the purified protein band was showing a shift to lower molecular weight than its original (where all other samples are in the same height). My protein molecular weight is ~45KDa. But after purification, i could see a band at ~30KDa. Could some one tell me the possible reason?
Do you have an antibody for your protein (or the His tag) to perform a western blot and confirm the presence of your protein on the gel? That would help to tell you if you're even looking at the right protein.
Thank you all for ur valuable suggestions. Unfortunately, we neither have antibody for western blotting nor mass spec facility. For the un-induced, induced, cell lysate and supernatant samples, i could see band at proper molecular weight (i didnt load pellet), but not after purification. So i suspect, there is problem in purification.
Since you do his-tag you do a western with 'anti his-tag Ab'. They are not very expensive and it could save you a huge amount of time...
Yoram
How about the flowthrough fraction? Are you sure your target protein was bound to the resin?
Please note, that several proteins of E. coli will be co-purified by IMAC, for example RplB with a molecular weight of 29.86 kDA!
Nature Methods, 2008, Vol 5, p 135, Review: Protein production and purification
I made this observation as well. At this time, I was not aware of the reference given above, and checked the co-purified protein by mass spectroscopy and identified an E. coli protein with several His-taqs nearby in its amino acid sequence.
This is normal if your protein is a membrane protein.
A. Rath, M. Glibowicka, V.G. Nadeau, G. Chen, C.M. Deber
Detergent binding explains anomalous SDS–PAGE migration of membrane proteins
Proc. Natl. Acad. Sci. USA, 106 (2009), pp. 1760–1765
How about the pI value of your target protein?
It's possible that if your target protein's pI is very low, then band shift may occur.
or flexible C-term region may be degraded.
So try the secondary structure prediction, and try the mass analysis of the target protein.
are you using protease inhibitors in your lysate? If not, your protein could be degraded by proteases...
Hydrophobicity and charge of your protein are the main factors that influence binding of SDS and therefore mobility during SDS-PAGE.
More details can be found here:
Article Global Analysis of Gel Mobility of Proteins and Its Use in T...
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