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His tag protein purification


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#1 freakyartemis

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Posted 13 July 2009 - 06:46 AM

I wanna purify a protein that is in vitro translated with the TNT Kit from Promega. I thought about Ni-NTA purification or magnetic Ni-NTA purification, but the protocol says that reagents such as amino acids are not recommended. My protein of interest is in vitro translated with the help of rabbit reticulocyte lysate where I add amino acids. Is there any way to purify the protein where the amino acids are not interfering the purification? Or is there a way to destroy the amino acids in the lysate, but not my protein??

#2 mdfenko

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Posted 13 July 2009 - 06:57 AM

you can dialyze the amino acids away from the protein solution

or

you can buffer exchange on gel filtration (sephadex g-25).
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#3 freakyartemis

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Posted 13 July 2009 - 07:05 AM

you can dialyze the amino acids away from the protein solution

or

you can buffer exchange on gel filtration (sephadex g-25).



Thanks for your quick answer!!!

Is that also possible with really low volumes? I have less than 1ml of lysate containing my protein...

#4 mdfenko

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Posted 13 July 2009 - 10:47 AM

low volumes are not a problem. dialysis can be done on sub-ml samples, we use narrow tubing, others may use a low volume slide-a-lyzer (pierce).

lower volumes are better for gel filtration columns. you will get smaller peak widths (and final volumes).
talent does what it can
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