I am using OPD tablets from Sigma as a substrate for my standard sandwich ELISA kits with HRP. I stop the reaction using 3M H2SO4 as described in the data sheet and the plate is then measured at 492 nm accordingly. This far I have used a microplate reader with a 630 nm filter as a reference wavelength and subtracted these OD values from the ones obtained at 492 nm. But now I am only able to access an instrument with a 600 nm filter instead of 630 nm. Is that sufficient or should I rather buy a new filter or not use any correction at all?
I don't think the 630 nm measurement is necessary. The protocols I read didn't mention it.
Dear
The measurement wavelength can be ranged 450nm to 620 nm.
Regards
I know this is quite late, but if still relevant you might find this paper useful: https://openi.nlm.nih.gov/detailedresult.php?img=PMC3003642_1746-4811-6-25-6&req=4
see Figure 6 A where absorbance at 600 nm to 630+ nm seems to be pretty similar.
BR
I am using OPD (p9029) peroxidase substrate in powder form. Does it matter to use in powder form or in tablet form? and what is preferable solvent for OPD????
It doesn't matter whether you use tablets or powder, but you should be aware that some tablet products should be dissolved in water (because the buffer is also contained in the tablet) and others should be dissolved in buffer. Check with the manufacturer. The powder should be dissolved in buffer, such as PBS.
Read the absorbance at 490 nm after quenching with acid, or at 450 nm if read continuously without quenching.
Thank you so much Adam B Shapiro. I need some more information please. Can we dissolve OPD powder in DMSO? and it should be freshly prepared every time or we can keep it in refrigerator?
I don't know about its stability once dissolved. I am confident that it will dissolve in DMSO.
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