Hi Amala, Depending on the resin you used there may be a conflict with BME and also 50 mM TRIS may be too much at the pH 7.5. Easy to test: use HEPES pH 7.5 and TCEP instead of BME, or use 30 mM TRIS at pH 8.0
Alternatively your protein is aggregated and does not bind well... Artem - Cosmic Cats approve of this message On Mon, Aug 13, 2018 at 9:49 AM, amala mathimaran <amalat...@gmail.com> wrote: > Dear All > > I am working with HIS – tag protein in N-terminal (hexa histidine). The > protein from Prokaryotic origin cloned into pET30a+ vector and expressed in > *E.coli > *BL21 cells. The expression was good. I am trying to purify a protein > using Ni-affinity column equilibrate with Buffer A 50mM Tris pH7.5, 50mM > NaCl, 15mM imidazole, 3mM BME, 5%glycerol and eluted with Buffer B ( same > as buffer A but 250mM imidazole). I eluted the protein in step wise > gradiant. the protein was less binded in Ni affinity IMAC column because > eluted fraction contain less amount and the protein remain present in the > Flow through. Can any one suggest how to increase the binding affinity of > the protein and how to purify the protein. The protein PI was 6.33 > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
