Dear Amala Please increase NaCl concentration to 200mM from 50mM. That can help you out by increasing affinity of your protein to bead and will delay the elution time.
On Mon, Aug 13, 2018, 7:20 PM 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
