This should give you everything you need to know about the column http://www.sigmaaldrich.com/technical-documents/protocols/biology/roche/complete-his-tag-purification-column.html
I'd definitely say load all of your lysate, possibly run it through twice if you really want to. If you're worried about it not binding run it at a slower flow rate. You would have to have a lot of expression from one litre of media to saturate the column. Not sure what exactly you mean by retention time but the protein should stay there indefinitely until you elute it. Cheers, Sam [http://www.sigmaaldrich.com/etc/designs/sigma-aldrich/images/sial-logo-sharing.png]<http://www.sigmaaldrich.com/technical-documents/protocols/biology/roche/complete-his-tag-purification-column.html> cOmplete™ His-Tag Purification Column Protocol ...<http://www.sigmaaldrich.com/technical-documents/protocols/biology/roche/complete-his-tag-purification-column.html> www.sigmaaldrich.com Protocol. Purification Protocols. cOmplete His-Tag Purification Columns are compatible with automated chromatography systems such as ÄKTAexplorer. ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Sundaram <maradnusmar...@gmail.com> Sent: 01 February 2017 09:24:09 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Manual His-Tag Purification Hello , It's an off topic question. I'm planning to do manual purification a 6 his tagged protein of size around 20kDa from 1L E.coli culture using COHISC-RO Roche cOmplete™ His-Tag Purification Column. Can I get some advice regarding the lysate loading volume and retention time. This is the first time I going to use this column and I have no idea about my protein yield from 1L culture. Sorry if I spammed your inbox. Thanks! Yours Sincerely, Sundaram.S
