We have got a recombinant protein which consist of target molecule and EGFP. However, the Mw of this fused protein is two times bigger than it was predicted. We are wondering if it could be a protein dimer or interaction with other cellular protein?
Hi,
How did you deduce your findings that your protein could form a dimer? Through SDS PAGE (denaturing)? gel filtration?
To look for dimer formation, you could try cross linking experiments.
Tell us more about what the results indicate a dimer: MS, BN electroforesis, size exclusion chromatography and other...
Hi there,
Dimer formation is an acceptable hypothesis. MS analysis of the content of the object would be more definitive. Analysis of the monomer/dimer dynamics (if monomer can be purified as such) would be a good point too. If dimer is actually occuring and as eGFP has a weak tendency to dimerize on its own, the next issue would be to determine what is the factor for fusion dimerization (eGFP or target protein).
Merci Dominique! Unfortunately, MS is not available. The results have been achieved using SDS-PAGE and Immunoblot. I wonder if I run electroforesis in reduced and non-reduces conditions do I see the difference in case of dimerized protein?
So it's a covalent dimer... Under reducing or non reducing condition?
Surprising! Are you sure your reducing condition was enough to ensure the disruption of all disulfides in the sample? I don't know any other covalent link occuring between polypeptides...
Well we use BioRad sample buffer, but don't add B-merc ( non-reducing).
Does this sample buffer contain reducing agent or not?
If not, it is worth repeating the SDS/PAGE and compare samples with and without reducing agent.
If yes, make sure your buffer is not too old (reducing agent tends to oxidize with time) or better add some more reducing agent in it.
If you still don't see any difference at the end, it means there is a covalent crosslinking between monomers (the only I know actually is the formation of isopeptidic bond between lysine side chain and Asp/Glu side chain...
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