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
signature.asc
Description: This is a digitally signed message part.
