Elution from PD-10 column - (Nov/18/2007 )

Hi. I have a purified protein in a high salt solution that I needed to desalt. I used the PD-10 desalting column. I washed it thoroughly with 100mL of ddH2O, then added my protein (2.5mL final volume). I collected the initial run through. Then I added 3.5mL PBS to elute. I checked concentration, but it was low, so I concentrated the elute down to a little under 1mL. Again, no readable protein concentration. I ran about 7mL ddH2O through the column, again collecting the eluted sample, concentrated that, but saw no readable protein concentration. I just finished a silver stain and saw nothing.

So my sample is still on the column, but what can I do to get it off? Does anyone have any suggestions or buffer recommendations? Any help would be greatly appreciated.

I was working with someone who said to wash with ddH2O. In the instructions, they used ddH20 to wash the columns for their BSA sample. I probably should have gone with PBS all the way or just eluted with water.

How did you concentrate your sample?
if you concentrated on an membrane, you have to know that you can lose quite a lot of proteins on a membrane.

That´s true, spin concentrating devices bind lot of protein. Are you able to boil a column resin sample, to check wether your protein is really still there?

Miguel

I think you first need to equilibrate your column with some buffer, or with the same buffer in which you are going to elute your protein. In my lab we also wash it with water first but then we wash it with the relevent buffer, then we load 2.5ml protein as you said and the elute in 3.5ml buffer. So is there any possiblity that your protein didn't bind to the column ? I mean I am just guessing . why don't you load your flow through on the gel and check?? I think it may be a possiblity.

may be your protein of interest was degraded by the protease

Ionic strength could be important for your protein remain solublized. Dialysis your protein agaisnt DW can tell you if it is the case or not. So equlibrate the column with buffer as suggested by many posts should be tested.

The other issue would be the concentation of the protein sample, make sure it is not too diluted or the protein can get absorbed non-specifically.

Thr PD-10 column typically takes less than 5 min to complete, so proteolytic event will not be a major factor here.

Thanks everyone. I appreciate the information. I used Amicon to concentrate my elution. I concentrated and checked the initial flow-through and the initial elution with a silver stain, and my protein wasn`t there. I finally found a weak band at about the 4th wash. I guess the bottom line is to elute with the same buffer as the wash. Does anyone have any other suggestions for desalting a protein sample, particularly when working with a very small amount of protein?