Lost protein after gel filtration - Gel Filtration (Oct/08/2010 )

Hi,
I m running my protein (importin alpha) over S200analytical column and nothing comes out of the column. I am injecting 45µg protein in a volume of 50µL into 100µL loop and empty the loop with 200µL. I thought that there was precipitate in the column and perhaps my protein is just sticking to that and therefore I washed the column enough with 1M NaOH and re run but the result was still disappointing. Any ideas about what might be wrong and what can I do about it? I run my protein on SDS PAGE and it is nicely seen there, so no degradation I believe. Any help is higly appreciated, thanks

the protein may be aggregated. this may prevent it from eluting. it may be aggregated before injection or it may be aggregating in the column.

if it is aggregating in the column then you may have to adjust or change your buffer (ionic strength, pH, additives, etc).

Hey, thanks for the reply. If it aggregates, then isnt it supposed to come in the void volume? Do u think it is more likely to get stuck to the column if it aggregates and it never comes due to that? What should I do to prevent aggregation? 5% glycerol would be sufficient?

if you dont mind me asking what is the buffer you are using. does it require nacl. I do not have experience in S200 but we used superdex 75 prep grade and we used to have same problem using plaing buffer but when we aded 0.15 - 0.2 M Nacl in the same buffer we got the protein elution. dont know of this information is of any use to it.
all the best

1324409 on Fri Oct 8 13:28:40 2010 said:

You are injecting a very small amount of protein. It could be sticking non-specifically to the superdex. I would certainly use > 100 mM salt in the elution buffer. In the past, when I was purifying low amounts of protein (cytokines) I had to include a small amount of non-ionic detergent (0.02% tween20) to prevent adsorption to the column/tubing. The tween does not cause problems with A280 and in non-denaturing.
Hope this helps

The buffer I m running with is 20mM Hepes and 110mM KAc buffer. When I purify this protein, I use this buffer to run over S200prep column at the last step. And it comes nicely at the expected size. So salt concentration should be no problem. I was also afraid of amounts but when I apply similar amounts of another protein, it comes out. I decided to load 1mg anyways and see what happens. I should see it in an hour Thanks to everybody for the comments.

1324409 on Fri Oct 8 19:23:56 2010 said:

it could become insoluble and get trapped in the column.

glycerol should have no effect on solubility, ethylene glycol might.

you need to ensure that your protein is stable and soluble in the buffer that you are using to develop your column.