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Query regarding Fluorescent based assay


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#1 SureshVM

SureshVM

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Posted 19 May 2010 - 09:43 PM

Dear All,

We are performing a fluorescence based assay to screen for inhibitor compounds of our enzyme and ultimately crystallize the enzyme along with inhibitor.

We see that some of our compounds are autofluorescent and thus are effecting fluorescence. In such a case we make a compound blank (assay buffer+ compound) and subtract it from test (enzyme + compound) but still we see the values of test much higher compared to control (only enzyme). In this case can we bring down the values of compound balnk and test by a factor which brings the value of compound blank to the level of blank and compare the values of resultant test value with that of control after subtracting the respective blanks.

For example we have the following arbitrary fluorescent readings (AFU) for one compound:

Blank (70342) Control (163243) Compound balnk (1865830) Test (2020455)

Since compound blank / blank ie (1865830)/(70342) = 26.52 can we do a normalization of compound blank and test values by a factor 26.52, which brings teh value of compound blank to teh level of blank and get the values (1865830)/26.52 = 70355 and 2020455/26.52 = 76186 and say the compound is inhibitor by comparing this test value with the value of control after subtracting teh values of respective blank.

Thank you in advance,
Suresh




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