Well. I am trying to purifiy an enzyme using blue agarose affinity beads. The enzymatic activity in the crud extract is 45 units/ml. When i incubated 2ml of crude extract with 5 ml of packed volume of beads and took out 0 Hrs fraction immidiately the activity became 20 units/ml. Now i am trying to elute the enzyme from the beads, but it is not working even using 10 mM of NADH as an elution buffer. I couldn't figure it out where is the enzyme has gone. Is it degraded or tightly bound to the beads?
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