Optimising an Enzyme Assay

[Monique]

I am currently busy with enzyme assays (well, trying to be): This is what I am doing, but its not working...

 

I induce mouse hepatoma cells with ethanol and a test compound. The test compound is supposed to cause the transcription of my enzyme which I am supposed to measure the levels of after cell lysis, freezing and thawing. However, after measuring the OD, there seems to be NO significant difference between ethanol and test compound induction, i.e. I generated two identical looking curves. This is so frustrating.

 

 

[chantel.bok]

It's likely that there is something wrong with your test compound, otherwise is it absolutely certain that this particular compound causes expression of the enzyme?

[Monique]

It's likely that there is something wrong with your test compound, otherwise is it absolutely certain that this particular compound causes expression of the enzyme?

 

 

Well, I'd like to think there is something wrong with the test compound - but yes - it has been shown previously. I am just having a hard time reproducing previous results. he other probem is - the levels of enzyme expressed is SO low ... its almost basal !

[chantel.bok]

Well, I'd like to think there is something wrong with the test compound - but yes - it has been shown previously. I am just having a hard time reproducing previous results. he other probem is - the levels of enzyme expressed is SO low ... its almost basal !

 

 

Have the results been 'reproduced' before - more than one reference? If you are doing the same thing as was done before (exactly), you should be getting the same results. Otherwise, there has to be something wrong with the compound. You are using the same concentrations and everything?

[CharonY]

First is always to troubleshoot the technical aspects (including lysis efficiency, measurement time etc.). After that you have to put some thoughts is whether there may be differences in you approach (e.g. cell line maintenance etc.). The description is insufficient to give detailed advice. However, how did you measure the enzyme expression? ELISA?Also I am confused on whether you measure enzyme activity or enzyme expression.

Edited September 2, 2010 by CharonY