hi everyone

so yesterday i posted a question in Biochemistry re: a nonspecific cleavage product i was seeing in an activity assay for abasic endonuclease 1.

i was looking at the coommassie gel of the purification, and notice a very dark band in all the imidazole fractions (i use a gradient elution) that is much smaller than my His-tagged protein. i used a 20mM imidazole wash buffer, and the went directly to a 150 mM imidazole elution fraction, 200 mM, etc. through to 1 M. the contaminant protein seems to be there in all these fractions, reducing considerably in the 1M.

my idea is to now purify the protein off the column using the 20 mM wash buffer, but then using a 70-80 mM imidazole fraction a few times to try to get this other protein off the column, then continue to a 200 mM elution for my protein. think this will work? thanks!

the co-eluted polypeptide could be a truncated form of the specific expression which may also carries the His-tag (could be verified with an anti-His-tag Ab); in this case you may not discriminate elution by washing or differential elution; think if you may live with this contamination if it is only a co-epression product to a minor extent