Hello Amala, Usually Ni-NTA won't have this kind of problem of binding. Probably your protein is have interaction with hexagon his tag which is affecting its affinity towards beads. You can try putting tag in C- terminal end of the protein.
On Mon, 13 Aug 2018, 8:02 pm Artem Evdokimov, <artem.evdoki...@gmail.com> wrote: > 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 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
