[Protein-analysis] Re: ELISA HELP NEEDED Cant Get anything to Work

[Allison]

Barbara wrote:

> I'm trying to develop 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 posted initially on the
> wrongforum I think.
> 
> 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.
> 
> 

If your first Ab is RABBIT anti mouse and your second Ab is goat 
ANTI-RABBIT then all you will detect is the coating primary Ab.

A better series would be:

Rabbit anti-ENAC
blocking protein
lysate
Mouse anti-ENAC
Enzyme-conjugated goat anti-mouse IgG

Whatever antibody you decide to use it is important to keep track of the 
animal it was made in as well as its specificity.

I have used the Nunc plates for ELISAs and I find that they work well. NAYY

HTH
Allison




> 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
>