Thanks Mark and for another off-board reply. My protein need only 20-25 mM NaCl but I include 500 mM NaCl for nickel affinity to avoid electrostatic interactions. 10 mM imidazole to avoid contaminant proteins.
I have tried to put N-terminus tag instead of C-terminus but it does not get exported into the priplasm. I will try omittig imidazole. Should I decrease NaCl to 20 mM also? On Fri, Jun 3, 2011 at 7:26 PM, Mark Nance <[email protected]> wrote: > Hi Matt, > > With that much NaCl, you can probably omit the imidizole during the loading > and wash steps. One of my proteins requires 500 mM NaCl, and even though it > has a 10-histidine tag it elutes at (or before) 50 mM imidizole. If you > still have your non-bound protein, you can dialyze/dilute out the imidizole > and run it back over the column. > > - Mark > > > > Message: 1 >> Date: Fri, 3 Jun 2011 11:57:40 +0100 >> From: mnr mnr <[email protected]> >> Subject: Nickel affinity purification >> To: [email protected] >> Message-ID: <[email protected]> >> Content-Type: text/plain; charset=ISO-8859-1 >> >> Hi. >> >> My protein contains 6 histidine residues for His tag purification. Less >> than >> a mg of protein is binding whereas therest about 10 mg don't. Both bound >> and >> unbounds have his tag on western blot. >> >> I am using 10 ml of nickel sepharose from GE in 50 mM Tris pH 8, 500 mM >> NaCl >> and 10 mM imidazole in sample and equilibrate buffer. Protein elutes at >> 200 >> mM imidazole. Is there any way to increase the affinity of protein >> binding? >> >> Thank you. >> >> Matt >> > > _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
