Dear Justin

Do you need high purity enzyme for biochemical caracterization or an enriched extract? What do you need it for (enzymatic assay or immunoassays)? Please give more information. 

Dear Sir,

Thank you for your kind response.

This enzyme is purely for an enzymatic reaction, primarily for an enzymatic inhibition study (I.e. Determination of enzyme kinetics etc.) The papers I read merely used lysates / tissue homogenized but I am afraid these may contain other enzymes that may affect the reaction. What grade of enzyme should I use and how should I isolate them?

For your advice, please.

Warmest regards,

Justin

Purifying an active native enzyme can be quiet troublesome since you don't have a affinity tag. It would involve multiple chromatography techniques and your yield would be very low (since you are working with cell extracts), that is why most people use lysates and homogenized tissues.

I agree with Sebastian. I believe you are trying to investigate if the enzyme from cancerous cell has different characteristics (Km, Kcat, Ki) when compared with a normal cell line. Since purification will be very difficult you could try to analyze comparatively using cell lysate from PC3 and normal prostate cell line. If you wish to try it, you could attempt to immunoprecipitate the enzyme from PC3 and perform your enzymatic assay.

Dear Sirs, thank you for giving me insights into my experiment and responding to my enquiry.

Actually my assay requires me to screen some compounds for 5a-reductase inhibition activity. Would you foresee any potential problems if I were to incubate my drugs directly with the prostate lysates (e.g. PC-3 cell lysates)?

For your kind advice, please.

Thank you.

Dear Justin

The only two minor side effects that I can foresee are:

1. The cells could already produce a normal inhibitor that could compete or intensify the effect of your compounds (I don't know if they do but it's a possibility). You could try to avoid this by comparing how your compouds act on normal AND cancerous cells. You could also try to enrich your extract using only one chromatography step like ion exchange or size exclusion. If you do this "clearing" you could eliminate a possible natural inhibitor.

2. Your assay could detect the activity of other reductase. I don't know the specificity of your assay (Does it work only for 5alpha or the substrate can be processed by different enzymes?). In this case you could change your question and investigate total reductase inhibition OR maybe try to knockdown each one of them (I believe there are 3 isoenzymes but I'm not sure) using siRNA.