[sani]

hello!

Has anyone used DAB (3,3' Diaminobenzidine tetrahydrochloride) used as a substrate to detect HRP in an ELISA? I already found out that the colour changes to brown, so it's a colourimetric dection and I can measure the Absorption @ 450 nm afterwards. If I have the powder only, what concentration do I need to make up and in what buffer and can I then just store aliquots @ -20C?

thanks, Sani

[pgenz]

I only have experience of HRP detection using a substrate such as TMB (3,3’,5,5’-tetramethylbenzidine) and hydrogen peroxide. I have used this previously and found it relatively easy to work with. Normally the range of addition is 50ul to 150ul but you must first carry out a calibration to determine what the desired max absorbance and min absorbance for detection is required. I used this in combination with a H2SO4 stop solution and read at 450nm.  Ideally the difference between the two values should be near 1 once optimized. I.e absorbance at 450nm at highest concentration on calibration scale; 1.45 lower range 0.45 etc.