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Lost protein after gel filtration


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6 replies to this topic

#1 1324409

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Posted 08 October 2010 - 05:28 AM

Hi,
I m running my protein (importin alpha) over S200analytical column and nothing comes out of the column. I am injecting 45痢 protein in a volume of 50無 into 100無 loop and empty the loop with 200無. 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

#2 mdfenko

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Posted 08 October 2010 - 08:03 AM

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).
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#3 1324409

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Posted 08 October 2010 - 11:23 AM

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?

#4 biotef

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Posted 08 October 2010 - 06:59 PM

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

#5 klinmed

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Posted 09 October 2010 - 02:16 AM

Hi,
I m running my protein (importin alpha) over S200analytical column and nothing comes out of the column. I am injecting 45痢 protein in a volume of 50無 into 100無 loop and empty the loop with 200無. 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

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

#6 1324409

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Posted 09 October 2010 - 08:04 AM

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.

#7 mdfenko

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Posted 12 October 2010 - 12:10 PM

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?


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.
talent does what it can
genius does what it must
i do what i get paid to do




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