Hi Sundaram, The binding capacity of this column is 40 mg/mL of resin so a 5mL column will hold a maximum of 200 mg of protein. If you run your cleared lysate on a gel you may be able to estimate how much protein there is. Our facility purifies a range of different proteins for investigators and our rule of thumb is not to load more than 1/3 of the column capacity so if your construct expresses more than 66.6 mg/L then you may want to batch load the protein. Without any knowledge of expression levels, I would recommend loading 1/10 of your cleared lysate then estimating total protein from your purified sample before loading the entire lysate.
Best, David -- David L. Blum, Ph.D. Director, Bioexpression and Fermentation Facility Department of Biochemistry and Molecular Biology University of Georgia 120 Green Street room A414A Athens, GA 30602 http://bff.uga.edu/ Skype: dlblum11 (706) 542-1035 (Office) On Wed, Feb 1, 2017 at 5:08 AM, Tim Gruene <tim.gru...@psi.ch> wrote: > Dear Sundaram S, > > during my PhD I used 4-7.5ml resin per l of culture, but I also had a large > yield of 60-100mg protein per litre. Try to use as little as possible - at > first trial check both flow-through and retentate by SDS-PAGE. (see. p. 40 > and > 54 of my thesis). > > My constructs would also bind greatly to Ni-IDA, but not at all to the much > more common Ni-NTA. > > When you expect low yields, and a protein that may be sensitive to the > immidazole concentration, you can also try Co instead of Ni. > > Best regards, > Tim > > On Wednesday 01 February 2017 02:54:09 PM Sundaram wrote: > > 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 > -- > -- > Paul Scherrer Institut > Dr. Tim Gruene > - persoenlich - > Principal Investigator > Biology and Chemistry > OFLC/102 > CH-5232 Villigen PSI > > Phone: +41 (0)56 310 5297 > > GPG Key ID = A46BEE1A > >
