measurement wavelength and reference wavelength - how to choose the suitable wavelength for colorimetric assay? (Jun/06/2008 )

Hi, can someone tell me what is reference wavelength? What is the difference between measurement wavelength and reference wavelength? Can I read the plate using ELISA plate reader without having a reference wavelength?

How do I determine the suitable measurement wavelength and reference wavelength to read the plate for a specific colorimetric assay?

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Reference wavelength is used for correction (normalization). Normally the protocol for commercial kit gives you the information for measurement and reference wavelength. For ELISA colorimetric assay, usually set 450 nm for measurement and 600 (or 620 nm) as reference wavelength.

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If the assay need to be read at 400 or 405 nm, what is the reference wavelength for it?

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it depends on the filter set you have, and also on the chromophore or pigment; the reference wavelength should not result in a prominent extinction (run a spectrum if possible)

you may try ~490 nm if you have no idea

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Does it mean the absorbance value read at 405 nm should be almost the same as the absorbace value read at 490nm? How u determine the reference wavelength? Read the plate at wavelength range from 400 nm to 700 nm? (whole wavelenth range of my ELISA plate reader)

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Can I run the measurement without reference wavelength? Just use 400 or 405nm as the measurement wavelength, and reference wavelength set as 0.

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