I am purifying a six-His tagged 50kDa hnRNP K protein from E coli cells using IMAC with Cobalt TALON resin. I am trying to send the proteins for crystallography and it requires a good quality of sample. My problem is that smaller bands are visible on the gel and I don't know how to get rid of them.
What might the problem and how can I fix it?
Can using a whole range of gradients (50, 60, 70, 80, 90, 100, 110mM, etc.), or adding imidazole to the wash buffer improve the specificity of the resin-bound proteins?
Attached is one sample of my purification on an SDS-PAGE gel. This was done after growing E coli on 1 liter of liquid media. From left to right: protein standards, lysis buffer mix, resin flow-through, wash 1, wash 2, wash 3, 50mM imidazole, 100mM, 200mM, 300mM.
Please Note that the wash buffer didn't elute out anything, and 100, 200, 300mM on the right side has a thick band with smaller contaminating proteins. I tried higher concentration of tris-HCl (but not NaCl)on the wash buffer but it did not improve the results for the washing step.
Thank you so much and appreciate your help!
P.S. Here is a short hand note of my reagents/protocol:
lysis buffer: 50MmTris-HCl (ph7.5), 10% glycerol, 0.5M NaCl;
Washing buffer: 50MmTris-HCl (ph7.5), 10% glycerol, 0.3M NaCl;
Elution buffer: 50MmTris-HCl (ph7.5), 10% glycerol, 0.3M NaCl, made 20ml each then add imidazole to final concentration of 50mM, 100mM, 200mM, 300mM to each.
Used 1L LB+drug to grow E coli up to OD600 of 0.6. Then induced the recombinant protein on a plasmid vector using IPTG for 4 hours 37C. Centrifuged and collected cell pellets. Went to 4C room, added lysis buffer with PMSF, sonicated, centrifuged, obtained supernatants, poured it onto cobalt-resin,
washed three times, and collected using imidazole elution buffer. SDS gel.