Hello there, I'm new to this forum.
I'm currently working on a recombinant protein with a C-term hexa His tag. When I open my cell cultures I got like 5% in the soluble fraction, the rest remain aggregated in the pellet. I run a step of Nickel purification on a column and see 4 bands, 3 bands in addition to my recombinant protein. These additional bands have been identified as various e. coli proteins by mass spectrommetry. I collect fractions and run a step of anion-exchange, again I see the same pattern of contaminant e. coli proteins. Gel filtration indicatates a soluble complex of these proteins as they elute in the same fractions.
I have tried purification under denaturing conditions because I have so much protein in the pellet. The idea is to make purification easy and then refold.
I treat with Urea (8M) in all buffers etc. still I see this same pattern of 4 bands, I try with Guanidine HCl (6M), DTT (10mM), Triton X-100 (0.1%) and still I have these 4 bands.
I simply don't get why I can't separate more efficiently, and have these proteins and be stucked to my recombinant protein.