Hi,
I have extracted enzyme (PPO) in phosphate buffer and PVP (15g/L) and then centrifuge (12,000 g, 10 min).
This seems this concentration seem to inhibit the PPO. So, I want to remove it to conduct the enzymatic assay.
Any ideas to do that ?
Pass the enzymatic preparation though a PD-10 or a small (25 ml) Sephadex G10 column. The initial 2-3 fractions shall contain the protein of interest.
Thanks for the answer but I haven't Sephadex colums. Is there another way ?
Try to borrow about 5-6 grams of Sephadex G-10 or G-15 to pack a column in 10 ml syringe barrel. Alternatively, dialyse the enzyme preparation in 10 kDa dialysis bag and use the dialysate for enzyme assay.
Dear Anne,
The approaches stated by Prof. Shamsher are accurate. You can subject the sample to dialysis. However, I would like to know if you have extracted your enzyme without using PVP? If so, was there any loss in enzyme? Why don't you try using PVPP for the extraction. But, given that you don't have access to the column material, you may proceed with the dialysis suggestion, which Prof has given.
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