My task was to purify Glutathione–S- Transferase isolated from E.coli cells. Using the following protocol: cell lysis, ammonium sulphate precipitation followed by dialysis, gel filtration (Sephadex G50), CM-Cellulose chromatography and SDS PAGE. The enzyme was assayed for protein concentration and enzyme activity at each stage in the process to determine specific activities, yield and purity. I have given the
Activity Total vol, [Protein] , only yield and purity of untreated is given.
(U/ 100 µl), (ml), mg/ml
but not the:
Activity, Total activity and Specific activity
(U/ ml), (U), (U/mg protein)
My question is how to find Activity ??/
(U/ ml)
If I understood your question correctly, just multiply U/100ul by a factor of 10 to get U/ml. Since you know the protein concentration (U/ml), specific activity (U/mg) is easily calculated.
Your results show enzyme activity as Uints per 100 ul so to get Unit per ml multiply this by 10 if protein concentration is known per ml sample you let assume it 10mg per ml and your unit per ml enzyme is 1000 then specific activity will be 1000 divided by 10 which equil to 10unit per mg protein. Goog luck
I think that for the GST purification it is important to use the affinity chromatography with agarose-GSH or other similar column this approch is better than gel filtration or ammonium sulphate precipitation. Concerning the Enzymatic activity expression i can send you the formula that i use.
Specfic activity is equal to the ratio betwen the total enzyme activity and the total proein =U(international units) / mg protein. In enzymology the AS is not the most fiable criterium,it is better to determine the turn over =maolecular activity= kcat= Vm/enzyme concentration (s-1) or the caralytic efficiency of the reaction=Vm/Km. This last ratio can help you to choose the better substrate.
After purification the enzyme should be found pure tested by SDS PAGE or preferable by the determination of the N terminal sequence (only some aA).
i think we saw this or something similar as a test question, very practical (important) calculations. Dr. Sridhar answer is easy to understand..
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