I'm new on his-tagged protein purification. I'm working with a recombinant protein of 13.1 kDa expressed in Pichia and I'm trying to purify it with a Ni-NTA column according with Probond native protocol of invitrogen.
I run my Tricine-SDS-PAGE to analyse the sample after pass it trought the column, and the washing (I'm performing 6 washes with native washing buffer with imidazole 20 mM an pH 8.0), and the elutions (my elution buffer has 250 mM imidazole and pH 8.0).
The problem is that apparently I've a good recovery of my recombinant protein in the elutions but also got a little contamination with proteins of lower molecular weight. Usualy when I'm passing the sample trought the column, I work on it in a room at 4 C degrees.
Does anyone a suggestion to get better results?. I'm thinking on dialyzing my elutions against binding buffer and try to re-purify it because but I'm affraid of lose a lot of it and I must have my pure protein as soon as possible.
Does anyone experience something similar?
P.D. Excuse my bad english.