Purified protein (his-tagged) multiple bands on gel - all picked up by antibody - what could they be? (Sep/18/2006 )
Hi,
I am expressing a his-tagged protein in E. coli, and purifying it on beads. I routinely observe (under SDS denaturing conditions) several smaller bands which also show very strong expression. The smaller bands are picked up by both my protein specific antibody, and also by a polyhistidine antibody.
I thought that transcription would always proceed from the start codon to the stop codon in a vector. My his-tag is N-terminal so it is possible that I could be purifying shorter transcripts, although I don't see why this would happen with this protein and not others that I work with.
Someone suggested that it could be different glycosylated forms. I have found some papers which say that E. coli can glycosylate proteins, but is this true of E. coli that we use in labs? I am using M15 E. coli. And, would glycosylation cause different sized bands under denaturing conditions? It appears that proteins might not be fully denatured, as my protein naturally exists in trimers and these can be seen on my gels as well.
Does anyone have any suggestions as to what the smaller bands could be? I have attached a picture. My protein is ~40kDa, the trimer runs at ~90. The protein specific antibody picks up almost all of the proteins on the gel.
Any advice greatly appreciated 
hello,
odds are, the smaller bands you see result from premature termination of translation. they could also simply be co-purifying contaminants. to clear this up, you can blot some of your protein and probe w/ anti-His tag Ab (just make sure the Ab matches the number of His residues in your tag).
the bands you see are not likely to be glycosylated forms of your protein, as your MW shift would go up instead of down if they were glycosylated.
one way to get around some of the un-desired bands is to re-transform your expression line w/ fresh plasmid before each induction. for whatever reason, pET vectors and other His fusion vectors tend to produce these lower MW bands as the glycerol stock ages.
you can also try inducing at lower temperatures (30 or 20 C) and induce at lower IPTG concentrations.
alternatively, you can simply express your protein w/a C-term His tag, and that way you'll only recover the full-length product via Ni affinity purification.
good luck.
I am expressing a his-tagged protein in E. coli, and purifying it on beads. I routinely observe (under SDS denaturing conditions) several smaller bands which also show very strong expression. The smaller bands are picked up by both my protein specific antibody, and also by a polyhistidine antibody.
I thought that transcription would always proceed from the start codon to the stop codon in a vector. My his-tag is N-terminal so it is possible that I could be purifying shorter transcripts, although I don't see why this would happen with this protein and not others that I work with.
Someone suggested that it could be different glycosylated forms. I have found some papers which say that E. coli can glycosylate proteins, but is this true of E. coli that we use in labs? I am using M15 E. coli. And, would glycosylation cause different sized bands under denaturing conditions? It appears that proteins might not be fully denatured, as my protein naturally exists in trimers and these can be seen on my gels as well.
Does anyone have any suggestions as to what the smaller bands could be? I have attached a picture. My protein is ~40kDa, the trimer runs at ~90. The protein specific antibody picks up almost all of the proteins on the gel.
Any advice greatly appreciated
Could it be they are degradation products?