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Decrease in concentration after protein purification


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3 replies to this topic

#1 monica.rivera3

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Posted 11 January 2011 - 05:30 AM

Hi,

I produced a protein but when I purify it my concentration decrease from ~9mg/ml to 2mg/ml. Can somebody have any ideas about what's happening??

Thank you

#2 antibodymania

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Posted 11 January 2011 - 07:20 AM

How are you purifying your protein? ie. IP? spin columns?

In my experience with purifying antibodies, a 50-60% loss is not uncommon. Especially if the protein is charged (or sticky), it may stick to the paper (or purifying vessel) which contributes to the loss.

There are lots of factors that can contribute to loss, don't get discouraged. :)

#3 monica.rivera3

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Posted 11 January 2011 - 08:27 AM

I purify it by Ni-NTA, remove the His-tag and the pass it through the FPLC (SEC). I inject ~550ul of protein in the FPLC and obtain like 2.3 ml with a concentration of ~ .5mg/ml. Then I concentrate the sample using centrifugal concetrators until I obtain 1.36mg/ml in 250ul.

Thanks for your help!!! :)

#4 MyProteinBulliedMe

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Posted 11 January 2011 - 07:03 PM

Another possible factor is that the protein degraded, or the concentration on the first one is wrong, or the concentration of the last one is wrong.

How did you test for concentration for before and after concentration? After many trial and errors, my best and most accurate testing is Bradford. Nanodrop can be screwed by DNA/RNA, and FPLC is not so accurate.

Bsaed on my experience, I think the most likely cause is that the protein "Crashed" (coming out of solution) in the concentrator, which is very common if you protein is unstable.

Or when you take the liquid from concentrator, you didn't "pipet it up and down" to mix the protein stuck on the sides. This has happened to me as well.

To get accurate concentration after using concentrator is, you should pipet it up and down, take the liquid, centrifuge it max speed in cold (4C) for 1 minute, and take the supernatant. The pellet is your "crashed" protein (denatured) and your active protein should be in the supernatant.

Hope this help.

Edited by MyProteinBulliedMe, 11 January 2011 - 07:05 PM.





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