I am purifying a protein which is showing activity in sonicated supernatant but when i am purifying it using ion exchange, eluted fraction is losing the activity.I did dialysis also even then the activity is not coming where as i am having protein band at right position.
Maybe your enzyme needs a cofactor, or an auxiliary protein, which you loose during purification. Alternatively, your protein may be unstable: adding a stabilizing agent, like glycerol, might help. Looking up the literature about similar enzymes may give some clues.
I am doing CM sepharose with 20mM Acetate buffer pH6 + 10mM Nacl and elution is done with 1M NaCl gradient.
I am doing sonication in same buffer and activity is seen in sonicated sup.
OK, as stated by Pierre you must look up if the enzyme requires a cofactor.
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