Absorbance reading does not match spectra - (Sep/06/2010 )

Hi All

I am using a new substrate so after my assay I ran a spectra (200-800nm) to determine the optimal wavelengh. The spectra looked very good with the peak at 630nm close on 2 absorbance units. I then read the wells at a single wavelengh of 630nm but the readings were in the region of 0.4 Au. Does anyone know why this occurs? And what can I do about it?

Just to add, I read at various wavelengths after that (i.e. 600, 610, 620, 640, 650, 660, 670, 680, 690, 700) as I received the same results - all reading in the range of 0.4 Au.

Any advice on this will be gratefully accepted.

Many thanks,
B

You have to give more details, what you're writing looks impossible.
The readings from your sample looks as the didn't react, a other possibility is that the reactionproduct breaks down. I,m just guessing by lack of information.

The path length is different between the spectrophotometer and your platereader.