Re: [ccp4bb] Na acetate as purification buffer

[Roger Rowlett]

Classical protein purification by IEX, HIC, GEC, etc. is apparently a dying art. It is typically quite easy to purify proteins using non-affinity methods from overexpression mixtures using an AKTA system. (Gosh, in the old days we used to purify to homogeneity proteins with 0.1% abundance in a natural source--a 15% overexpression crude extract is trivial by comparison.) It is fairly quick to scout good step gradient conditions for partially purifying your protein by ion exchange by using a 1 mL or 5 mL ion-exchange column, then scale up to a 1.6x10 cm or 2.6x10 cm column, depending on your crude extract sample volume. (We maintain Q- and SP-sepharose columns in our lab for low- and high-pI proteins, respectively.) A secondary purification via hydrophobic interaction or even salt fractionation is typically sufficient to clean up well-overexpressed proteins. Desalting and polishing can be accomplished on a large (1.6x60 cm) gel exclusion column. We purify our current crop of proteins we are studying this way--no tags to remove later. Sometimes, the time saved by affinity purification on the front end is eaten up on the back end with the necessity for purification tag removal. Don't be afraid to give the "old" methods a go. As Artem has pointed out, if your target protein has an unusual pI, it may essentially purify in one step (IEX) and you can then do a quick polish on GEC. Cheers, _______________________________________ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 3/1/2012 11:55 PM, Artem Evdokimov wrote: This pH is generally incompatible with Ni IMAC, sorry :) If you have a high pI your best bet is to employ ion exchange as primary capture, specifically SP resin or if you're really lucky - CM resin. There are only relatively few proteins in E. coli that bind to CM resin at pH 5 and virtually none (one-three) that will bind at pH 8. If your protein still binds, then you're good to go. Artem On Thu, Mar 1, 2012 at 10:49 PM, anita p <crystals...@gmail.com> wrote: Hi all, Has anyone used sodium acetate buffer pH (4-5) for purifying histag protein on Histrap column (AKTA) followed by SEC? My protein has a pI of 9. I tried pH7.4 but it has precipitation problems. While doing buffer screening using 24 well hanging drop I found that lower pI onces are clear, so just thinking can I use Na acetate at pH 5 for whole purification???  Thanks in advance  Anita