I am using GST-affinity tag to purify my protein. The standard protocol inculdes ~150mM NaCl in the GST buffer. In order to remove the DNA (maybe interact with my protein), I want to increase the concentration of NaCl(~1M). Is this problematic?
Ya we use 1M NaCl in lysis and 0.5M NaCl in washing buffer to avoid those contaminating DNAs and its consequencses. Have a look at the reference:
http://www.nature.com/nsmb/journal/v19/n10/full/nsmb.2378.html
That's quite a lot of salt, not sure but it might interfere with the GST interaction with the purification matrix. What about just treating your lysate with DNase? I do it often in bacterial extracts in order to get rid of DNA without sonicating or in cell extracts for IPs of chromatin proteins
Thank you Daniel. I already used DNase. DNA still exists since DNase may only cut it into small fragments. These fragments were also troublesome.
Ah ok.
I know that at least 500mM salt is probably ok, a lot of people use it for washing, so for binding might work too. There also a bunch of methods to get rid of DNA, namely using PEI, I have no experience with those. Refence here: http://wolfson.huji.ac.il/purification/tagproteinpurif/dna.html
Ya we use 1M NaCl in lysis and 0.5M NaCl in washing buffer to avoid those contaminating DNAs and its consequencses. Have a look at the reference:
http://www.nature.com/nsmb/journal/v19/n10/full/nsmb.2378.html
I have used 500mM of NaCl for GST affinity purification. I have never tested higher concentration. So at least until 500mM it should work.
I have used up to 1M NaCl for GST (and polyhistidine) preps with no problems.
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