IEX Elution pattern different

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msrcmurthy
msrcmurthy's picture
IEX Elution pattern different

Dear All,

In my Protein purification I am employing Source 15Q, anion exchange beads, to purify my protein.
(a) when I start with fresh beads, my protein is getting separated from the impurities and it is working well,
(b) when I want to do another batch of purification on the same beads my protein is not getting separated from the impurities.
Remember the beads are thoroughly done cleaning in practice (CIP) and regenerated according to the manufacturer's recommendations, but still the protein is not getting separated from the impurities.but I use fresh batch again separation is excellent. under same buffer conditions, gradient etc

Can anybody help me in sorting this problem

thanks in advance,
Dr.Murthy

jangy
jangy's picture
sounds like some non specific

sounds like some non specific binding going on. usually, the resin backbone itself is pretty "sticky", so it can actually bind some interfering molecules onto the resin. You may try different regeneration, sample prep, mobile phase, etc.