ENZYME ASSAY WITH PLANT CRUDE EXTRACT - (Sep/13/2008 )
Hi,
I working on enzyme assay to evaluate the inhibition of plant extracts on Phase I and II drug metabolizing enzyme. The enzyme source is from male rat liver, produce by centrifugation to obtain the cytosolic fraction and microsomal fraction.
The result I got is very weird. The result is not concentration dependent. At lower plant extract concentration the enzyme activity (GST) increase but x significant...then at a higher conc, it decrease and significant but at more higher conc the enz act. increase again ...only at 500 ug/ml and above conc. the GST enzyme act decreased significantly
My advisor suggest that the color of my crude extract intefere with the result. Is there any method to bleach the color of my plant extarct?
My friend on the other hand said that maybe the other compound in the crude extarct intefere with the result, so i should use pure compound instead as my positivi control using tannic acid gave a very good inhibition pattern.
Any of u guys here have idea on whta shoul I do to solved this problem
TQ
I working on enzyme assay to evaluate the inhibition of plant extracts on Phase I and II drug metabolizing enzyme. The enzyme source is from male rat liver, produce by centrifugation to obtain the cytosolic fraction and microsomal fraction.
The result I got is very weird. The result is not concentration dependent. At lower plant extract concentration the enzyme activity (GST) increase but x significant...then at a higher conc, it decrease and significant but at more higher conc the enz act. increase again ...only at 500 ug/ml and above conc. the GST enzyme act decreased significantly
My advisor suggest that the color of my crude extract intefere with the result. Is there any method to bleach the color of my plant extarct?
My friend on the other hand said that maybe the other compound in the crude extarct intefere with the result, so i should use pure compound instead as my positivi control using tannic acid gave a very good inhibition pattern.
Any of u guys here have idea on whta shoul I do to solved this problem
TQ
Hi, I have no idea on how to bleach the color of extract...you are using 96 well plate (microplate reader) or cuvettes (spectrophotometer) for your measurement?
Actually I want to ask whether u do protein quantification after u lysed the cells and collect the supernatant. I am trying to do tyrosinase enzymatic assay. Enzyme tyrosinase need to be extracted from the melanoma cells. Then I need to do protein quantification so that I use the equal amount of protein for the assay each time. Do u have any related protocol about cell lysis and protein quantification from the supernatant collected?
Hope can get a detailed protocol if you have. Thanks!