Hi All!
"Thorsten Schmidt" <Thorsten.Schmidt at rz.ruhr-uni-bochum.de> wrote:
>I have big problems with ELISA.
>The main problem is that the color does not develop satisfactorally.
(Other Stuff Deleted...)
>Some of my experiments might have shown that H2O2 might be an important
>factor for color development.
Yes, the H2O2 is very important, as it is one of the two substrates
needed (obligately) for the HRP reaction.
if your 30% H2O2 is old (or even if it is relatively new), it may be
significantly degraded, such that your final concentration of H2O2 is
insufficient to carry out a good reaction and develop the color you
expect. Your OPD solution should yield a final [H2O2]=0.045%;
parenthetically, we usually use 0.02% final, and it works fine. You can
check the quality of your H2O2 by doing a 1:100 dilution with pure water
and read the absorbance (vs. water) at 250 nm; the extinction coefficient
of H2O2 at this wavelength is 16.69 M^-1 cm^-1 or 7.18 (1%, 1cm). If it
is degraded, get new reagent, and your problems are likely fixed.
I hope this proves somewhat useful. Have a great day!
Best regards,
Shaun
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= Shaun D. Black, PhD | Internet address: shaun at uthct.edu =
= Dept. of Biochemistry | University of Texas Health Center, at Tyler =
= World Wide Web: http://pegasus.uthct.edu/UTHCT-Home/Welcome.html =
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