You are measuring fluorescence changes which are likely to be due to
compound binding by your enzyme.
In this case your blank must be your "compound" blank and no other.
Then you measure changes in fluorescence intensity and lambda max of
emission as you add your enzyme.
I do not know you system.
Note however that binding (1) might be non specific and (2) need not
correlate with enzyme inhibition.
Pr. Nadir T. Mrabet
Structural& Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax: +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet<at> medecine.uhp-nancy.fr
On 19/05/2010 08:48, suresh mattegunta wrote:
Dear All,
Sorry for the non crystallography related question
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
--
Suresh V Mattegunta MS
Senior Research Associate
Discovery Biology Division
Aptuit Laurus PVT LTD.
ICICI Knowledge Park
Hyderabad 500078 India
