Thanks everyone! Thanks Prof. David Blum. I will do batch load and check.
Yours Sincerely, Sundaram.S On Wed, Feb 1, 2017 at 9:06 PM, David Blum <dlb...@gmail.com> wrote: > 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 <070654%2021035> (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 >> >> >
