After purification, I am trying to concentrate the protein with ultrafiltration tube (Sartorius, Vivaspin-30 kDa) but i am losing the protein after uf. The protein is 54 kDa and its pI is 8.3, there are some non specific bands at like 30 kDa. I can neither remove the impurities nor concentrate my protein. I always check the protein after purification and uf with SDS-PAGE.
If anyone can offer advice on this matter, I'd be grateful. Thank you.
If your protein is not concentrating then perhaps it is a shape that allows it to pass through a 30kda pore size. It might be worth trying a smaller pore size membrane. You can concentrate protein by swelling sephadex or other size exclusion matrix which removes water but does not allow the protein to enter the matrix....very quick but you will lose some protein or freeze drying is an effective concentration method. Is it possible that your large protein is a dimer held together by SH bonds and removing stabilisers through the membrane causes monomerisation and loss of activity?
Using a 10kDa cut-off will ensure protein recovery if there is no non-specific binding or proteolytic cleavage. However, using a 30kDa application is a bit risky for 54kDa protein. To see where your protein is, load both retentate and filtrate into a PAGE gel after the UF process.
You can get answers at the end of this experiment such as;
Is it prone to be hydrolyzed during the process?
Is the cut-off value proper to hold your target in the retentate phase?
Does your target bind to surfaces or membranes?
As someone mentioned already, I will never use a MWCO membrane that is half the size of the protein. On the other hand, you can always lose protein due to non-specific binding to the membrane (no matter what the manufacturers say). Moreover, if your protein is sticky (a high number of hydrophobic patches on the surface), once one molecule is adsorbed and obstructs the pores, you can always expect that protein binding occurs due to the hydrophobic effect, you are essentially promoting protein-protein interactions due to the ultrafiltration system per se (crowding).
You don't mention if your protein is present in the filtrate or not. If yes then it is a question of too large cutoff. If it is neither in the filtrate or concentrate then it is probably building a gel on the membrane because of too high local concentration. In that case UF is not the way to go. Try to concentrate it by chromatography - ion exchange can often work because the resins generally have very high capacity.
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