Hi there,

Is the protein stable before NiNTA?

What are the buffer pH and composition?

According to the title of your post the volume of the eluate is very large (200mL for 34mg), is there any reason to work at very low protein concentration? Affinity chromatography presents the advantage of efficient elution in small volumes in order to avoid large dilution factors.

In a first move I would reduce the eluant volume in order to get something like 10mg/mL and then inject this directly onto SEC column to remove imidazole.

There are two issues here: removal of the imidazole and concentration of the protein. The first thing to do is decide whether either of these steps is necessary. Depending on what you plan to do with the protein, you might be able to store it in aliquots "as is" just by flash-freezing them.

I think you concluded that the first batch precipitated because it was stored in 200 imidazole, but maybe that was not the reason. The protein may be unstable, it may aggregate in high ionic strength buffer, it may not like being stored in soluble form in the cold.

Based on some of my previous experience with a multi-subunit protein that was unstable when stored as a solution in high salt in the cold, a good way to store it, somewhat surprisingly, was to to precipitate it with ammonium sulfate and store it at 4oC as an ammonium sulfate precipitate. In your case, that accomplishes both goals. When you spin down some of the precipitate and resuspend it, you can increase the concentration by resuspending it in a smaller volume. At the same time, you get rid of most of the imidazole, which remains in the ammonium sulfate supernatant. Then, before you use the resuspended protein, you exchange it into the desired buffer using a gel filtration spin column or regular gel filtration column to get rid of the salt. At this point, you may find the protein is more stable at room temperature than in the cold.

You could also try adding a stabilizing agent, such as glycerol, sucrose, or trehalose. Also, a little bit of non-ionic detergent may help if the protein has a tendency to aggregate.

By the way, you might do better with a 10,000 MWCO concentrator instead of 3000. It goes faster and is less likely to concentrate low-molecular solutes like imidazole.

@Adam Sir, thank u for ur suggestions

the protein is a subtilisin protease can i lyophilize it?

@dear Liger the buffer is sodium phosphate with a pH of 7.4

with 300mm NaCl

I would try to avoid phosphate buffer also, so going with extensive dyalisis versus an hepes or tris based buffer. if you have a so high volume, you could try also to test different conditions in parallel (using aliquots): low salt, high salt, reductants, detergents, arginine, glycerol, ... just to undestand which could be the best buffer before the concentration. good luck

If I were you I would try elute the protein in less volume, then dilute the eluant to < 100 mM imidazole since amicon concentrators cannot withstand high concentrations of imidazole, and also I would use 10 kDa MWCO, because your protein is 45 kDa. 

use centrifugation-based concentration methods and dilute a few times with your final buffer

Sigma sells subtilisin powder, so it should be possible to lyophilize it. You may want to dialyze out most of the salts first.

http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/subtilisin.html

@adam

Sir thanks a lot. i also had thought of lyophilizing it. thank u for ur concern

@Nan what would be the rpm? if u could suggest