On the DEAE, try lower ionic strength or slightly higher pH, try DEAE-Sepharose instead of cellulose, to get it to stick
Try a cation exchange column like carboxymethyl- or phospho- cellulose; at slightly acidic pH if necessary. eab Narayanan Ramasubbu wrote:
Hi all: I am trying to purify a protein that was expressed in a baculovirus system. The protein elutes in the flow through in a DE52 column. Further gel filtration gives an enriched protein but we see that some medium components tag along (weight and activity data do not match). Another mutant in the series also gives 30 mg of colorless fluffy material but again we never got this much protein for other mutants in the same series. We suspect that some "medium" is tagging. We tried other chromatography such as hydroxyapatite, activated charcoal, anion exchange etc but not "successful" in getting rid of the "impurity". Has anyone come across a protein that eluted in the flow through and if so, what is their experience? Did they notice such behavior? Your advice will be greatly appreciated. Thanks Subbu
