Dear all
I expressed my gene of interest in pET30a vector and Bl21 codon plus host (E.coli), the protein expressed successfully i check it in SDS-PAGE and by western blotting (Both with hyper immun sere & Anti his). But when i try to purify my protin is not binding to the olumn. I tried with both Native & denature form, pH from 8.0 & 7.5 and Lysis buffer with Imidazole and with out. Any one please help me to come of this problem.
Now I am planing to change the vector, there it really works, means if I change vector it really helps in purification.
Please help me.
Thank you soo much for your valuable suggestion. Here i have answered all your question please go through and help me out
Q.1) Yes i found a dark band in SDS-PAGE , its not in controls (non induced cell)
Q.2) In western blotting i use whole cell pellet against hyper immune sera and i use clear lysate after centrifugation against anti his antibody.
Q.3) I see my protein in lysate pass through fraction.
Q.4) As per the kit instruction i use 10 column valume of 0.5M NaOH and store my column in 30% ethonal.
Dear sir
For purification I use the Ni-NTA column Cat no: Cat No./ID: 30761 as per their instruction. but buffer composition are
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20 mM Tris-HCl, 0.1 M NaCl, 0.1 M EDTA, 8 M urea pH 7.5.
Refolding
Refolding of the bound protein is performed using a linear 6–0 M urea gradient, starting with the wash buffer above and finishing at one without urea.
20 mM Tris-HCl, 0.5 M NaCl, 0.1 M EDTA, 20 mM imidazole, pH 7.5.
Washing buffer:
20 mM Tris-HCl, 0.5 M NaCl, 0.1 M EDTA, 20 mM imidazole, pH 7.5.
Elution
Elute the refolded recombinant protein using a 10–20 ml 20 mM Tris-HCl, 50.0 mM NaCl, 0.1 M EDTA, 500 mM imidazole, pH 8.0.
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