ELISA HELP NEEDED Cant Get anything to Work

WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de)
Fri Mar 23 16:43:19 EST 2007


Hi,

You want to assay your proteins, not the antbodies, don't you? So You
could try this: coat your lysate. goat anti rabbit won't stick
anywhere except maybe a rabbit IgG.

If you can get antibodies against different epitopes of your protein,
you could try to set up a sandwich elisa as in the kit:

Coat eg with species 1 anti protein (epitope 1), block with BSA,
incubate with your lysate, wash, incubate with species 2 (not mouse in
your case) anti your protein (epitope 2) , wash again and then detect
the bound anti epitope 2 ab with anti species 2 (must be different
from 1 but can be mouse) coupled to reporter, eg peroxidase and then
produce colour.

ATPase maybe can go by liciferase assay in a luminometer. might be a
bit difficult to calibrate, as usually this assay is the other way
round (ATP), but assuming that ATP degradation is somewhat linear with
ATPase conc, it should work when you find the right dilution range.

Concerning biotinylation, probably endogenous biotin from your sample
is displacing the immobilized AB from the strep plates. You could try
chemical immobilizing with cycuric chloride or another bifunctional
reagent (see eg pierce or sigma catalog) on amino coated plates
instead.

Best regards,

Wo


On Mar 23, 4:49 pm, "Barbara" <blarr... from aol.com> wrote:
> I'm trying t odevelop an ELISA Assay to measure ENAC & ATPase in mouse
> cochlea lysates. I've been able to do so using NFkB, TNF & VEG kits.
> But there are no kits for these ab.
>
> I have tried coating the Nunc maxisporp plates ( at a couple of conc)
> with rabbit anit-mouse ENAC or rabbit anti-mouse ATPase then add my
> antigen (mouse lysate) followed by ALphatase goat anti rabbit IgG. It
> seems that nothing sticks. Given the small amount of lysate &
> variability coating wit hthat is not practical.
>
> I also tried biotynating the anti ENAC & ant ATPAse & then incubating
> with coated plates. Followed by blocking & then my mouse lysate & the
> nthe goat anti rabbit. Every thing is high except fo the ones without
> the biotinalyted AB initially added.
>
> Anyone have any thoughts or suggstions.
>
> Thanks
> BL




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