In a complete native conditions usually proteins do not appear as sharp band. smear like band is a common phenomenon, becuase without SDS the movement of protein do not follow the gel pore size/ molecular weight criterion. If your pure protein is 21 kDa and you get single band in SDS-PAGE, the high molecular weight band of same protein (pure fraction) in native condition suggest the polymeric condition of the pure protein (may be octamer?). you can see oligomeric condition in native PAGE easily. The most feasible will be to do the gel filtration of your protein. just inject 200-500µg of your protein in gel filtration analytical column (Superdex 200), or self packed sephacryl 200 and separate it. use standard molecular weight marker to calculate the molecular weight. confirm peaks by using native and SDS-PAGE to be sure that it is your protein.

I will also suggest you to do the seminative PAGE (Non-reducing conditions). Dont use beta-Mercaptoethanol in loading dye, and sample 1) dont heat protein sample before loading to gel, 2) heat 3-5 min at 95 degrees another sample before loading. Load these both samples in normal SDS-PAGE (may be 12%), dont make stacking gel, full resolving. This often shows very sharp oligomeric band, which you can compare with molecular size markers.

In a complete native conditions usually proteins do not appear as sharp band. smear like band is a common phenomenon, becuase without SDS the movement of protein do not follow the gel pore size/ molecular weight criterion. If your pure protein is 21 kDa and you get single band in SDS-PAGE, the high molecular weight band of same protein (pure fraction) in native condition suggest the polymeric condition of the pure protein (may be octamer?). you can see oligomeric condition in native PAGE easily. The most feasible will be to do the gel filtration of your protein. just inject 200-500µg of your protein in gel filtration analytical column (Superdex 200), or self packed sephacryl 200 and separate it. use standard molecular weight marker to calculate the molecular weight. confirm peaks by using native and SDS-PAGE to be sure that it is your protein.

I will also suggest you to do the seminative PAGE (Non-reducing conditions). Dont use beta-Mercaptoethanol in loading dye, and sample 1) dont heat protein sample before loading to gel, 2) heat 3-5 min at 95 degrees another sample before loading. Load these both samples in normal SDS-PAGE (may be 12%), dont make stacking gel, full resolving. This often shows very sharp oligomeric band, which you can compare with molecular size markers.

If you have a purified protein, I would suggest you to go for ‘Dynamic Light Scattering (DLS)'. The DLS will tell you what kind of complex your protein formed (dimer, oligomer etc.). Importantly, you can correlate your gel filtration (suggested above by Birendra Singh) and DLS results for further confirmation.

This reminds me of metallothionein - high zinc and high cysteine containing protein that forms polymers under oxidising conditions and when metals are removed. I'm not suggesting that your protein is MT becaiuse the MW is too high, but have you checked to see whether it naturally contains metals?

Thanks for your suggestions. My novel bioactive protein (N-terminal sequencing) is closely resembling the auxin-binding proteins, so common in plants and earlier reports suggests that it exists as a dimer with subunit molecular weight of 21,000. Auxin a phytohormone has a mass of 175kDa. I would definitely try to conduct seminative PAGE as suggested by Dr. Birendra and try to observe the sharp oligomeric band. I would appreciate more technical inputs to my scientific query. I would also like to know more about DLS as suggested by Dr. Pujara.

You can work with dynamic light scattering (DLS) that tells you how many populations (molecular sizes) of proteins are present in a solution. But if you have complex with some other protein then it will be only one peak, but if they are separate then you can see different peaks.

CAT gel electrophoresis is one of the best (in gel) option to find out exact Mr Wt of protein . Where CTAB detergent (cetyl trimethyl ammonium bromide) is used instead of SDS.