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Alkaline Phosphotase substrate for ELISAS - (Sep/07/2005 )

I've just done my first ever ELISA assay and it worked!!

However I'm a bit worried by the results. I'm using the Bio-Rad Alkaline phosphotase substrate kit (cat no:172-1063) and I got a very strong yellow colour in my treated wells. The colour seemed to keep developing for about an hour until I threw it away. Does this always happen? One of my dillutions shouldn't have worked, and didn't appear to at first, but the colour was very strong after only ten minutes. Do I need to stop the colour change (there are instructions given on how to in the kit) and if so after how long?



We use 50┬Ál 2N (=1.8M?=10%) Sulphuric Acid per well to stop our ELISA development. There's a colour change in the TMB (HRP-substrate) from blue to yellow when the sulphuric acid is added.

If you don't stop the ELISA or read it quickly eventually all your standards will plateau out and your standard curve won't be much use.

When I started doing ELISAs , one of the other post-docs suggested stopping the reaction when you can see a visible difference between your middle two standards.

Leave the plate until you see visible differences between your standards and then use the method suggested in the kit to stop the reaction. I guess a bit of trial and error might be involved.

Hope that helps.


The TMB substrate is stopped with acid and this will probably work for the alk phos as well-anything that radically changes the pH will stop the enzyme reaction but I don't know enough about the chemistry to know if the substrate is acid labile??? However I have always used 0.4M NaOH for my alkaline phosphatse assays and I use the same BioRad substrate as you (cat no 172-1063).

The colour will keep developing for approx 24 hours but true positives should be visible within the hour. If your negative controls are developing colour then the reaction should be stopped immediately.