in vitro enzyme assay - (Jul/21/2008 )
Hi,
I want to enquire on in vitro enzyme assay using subcellular fraction. It is normal to get inhibition result during enzyme assay but is it normal to get induction during the assay? How can i explain if i get induction. Do the result valid?
I want to enquire on in vitro enzyme assay using subcellular fraction. It is normal to get inhibition result during enzyme assay but is it normal to get induction during the assay? How can i explain if i get induction. Do the result valid?
Hi,
If we think naive (without any pre-conceived ideas), we can also get induction of an enzyme during in vitro assays. I have worked with enzymes that are induced by routine molecules like ATP, NADH etc. You mentioned about sub cellualr fractionation. I would think like: either something in the fractionation buffer (EDTA or something) or just concentrating a native molecule during fractionation could induce an enzyme in vitro. Without knowing what is the enzyme activity you are assaying for, it is hard to tell anything further. But, do not trash the results for the moment. Just record and save it until you have an explanation.
I do an experiment to observe GST specific activity in the presence of herb. I also do a positive control by using tannic acid, a known inhibitor for GST. At lower concentration of herb (less than 100 ug/ml), the activity is increase, and significant at concentration near 100ug/ml. But if I use higher concentrations > than 100 ug/ml, the activity is reduce compared to control. But the same pattern also occur if i substitute the herb with tannic acid. it will induce the enzyme activity at lower concentration and prominently inhibit after 10 ug/ml compared to control. There is somting wrong with the lowest concentration (0.01ug/ml,0.1 ug/ml)...
Jude, could u explain further what was happened?
TQ
I want to enquire on in vitro enzyme assay using subcellular fraction. It is normal to get inhibition result during enzyme assay but is it normal to get induction during the assay? How can i explain if i get induction. Do the result valid?
Hi,
If we think naive (without any pre-conceived ideas), we can also get induction of an enzyme during in vitro assays. I have worked with enzymes that are induced by routine molecules like ATP, NADH etc. You mentioned about sub cellualr fractionation. I would think like: either something in the fractionation buffer (EDTA or something) or just concentrating a native molecule during fractionation could induce an enzyme in vitro. Without knowing what is the enzyme activity you are assaying for, it is hard to tell anything further. But, do not trash the results for the moment. Just record and save it until you have an explanation.