Some papers say that this protein shows such behavior due to charge state distributions. I am not getting a proper explanation for the same. The protein is cytosolic, soluble when recombinantly expressed and exists as 80% and 20% dimer in SEC (this happens with this protein with other organisms too).
Please check the following.
1. Running conditions for SEC are native for the protein.
2. Does the protein require any metal ion for its stability?
3. It shows some expansion in the native conformation.
Saad, the running conditios are native, no metal is required.... now I think the protein shows not a true globularity in its native structure, and due to that this pseudo expansion is observed...
no, its a redox protein of mol wt around 26 kda and is coming at around 32-33 kda in S-200 and S-75 columns
Did you check the presence of essential cofactors required by the protein? Noncovalent association of these cofactors may affect the conformation.
Did you consider that it could be an intrinsically disordered protein? Try recording a far UV CD spectrum. Look at this:
The most common reasons for an earlier elution (apparently higher molecular weight) would be conformation or cofactor binding, mentioned earlier, or rapid dynamic monomer-oligomer equilibrium. You can check for the latter by running a few different concentrations to see if the behavior changes with concentration. This will also show up quite clearly in SEC-MALS as you'll see the molecular weight change both across the peak (due to concentration-dependent self-association) and as you change the injected concentration. See www.wyatt.com/Proteins under 'native oligomeric state' for more on that topic.
www.wyatt.com/Proteins
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