bijgh at zeus.bris.ac.uk (Jared Head) wrote:
>Hi,
>Thanks to everybody for the help on peroxidase/phosphatases. I thought
>I'd try you out on my next problem!
>I've just done a titration of secondary antibody against my antigen, to
>characterise the non-specific binding. When I added my colour substrate
>(TMB in DMSO/citrate buffer) some of the wells that contained no
>peroxidase conjugate gave a much larger colour change than the general
>background. Is this because I have somehow contaminated these wells with
>secondary antibody/conjugate, or is there another explanation?
>Thanks,
>Jared
In the absence of other information, the answer must be, probably.
Additionally, and this is especially true of ELISA's, don't begin to
panic until, checked what you physically did, and then you've repeated
the effect
a) with the same reagents
b) newly made-up reagents
...then panic :)
BTW we have switched to storing our TMB stock diluted in DMF rather
than DMSO, because its still liquid at 4 deg C (sometimes every minute
counts).
Pete
