ELISA Develop. Problems with Capture Sandwich Technique - (Mar/25/2007 )
I'm trying to develop an ELISA Assay to measure ENAC & ATPase (proteins) in mouse
lysates. I've been able to do so using NFkB, TNF & VEG kits. But there are no kits for these ab.
I only have a small amount of lysate/sample so coating it is not an option. So I believe the cature/ sandwich ELISA is the best.
I have tried coating the Nunc maxisporp plates (at a couple of conc) with rabbit anti-mouse ENAC or rabbit anti-mouse ATPase. Then add my antigen (mouse lysate) followed by alkalinephatase goat anti-rabbit IgG. I don't get any color.
I have also tried biotinynating the anti-ENAC & anti- ATPAse & then incubating with on Pierce coated strep. plates followed by blocking. Thinking that the would get around what I assume is the anti-enac or anti-atpase not sticking. Then figuring I'd just start my real asy from there.
Add the mouse lysate samples. Detect with the goat anti-rabbit. Every thing is high except for the ones without the biotinalyted AB initially added. this would suggest the 2 is just binding to the 1 AB.
Any useful thoughts or suggestions would be appreciated.
Thanks
lysates. I've been able to do so using NFkB, TNF & VEG kits. But there are no kits for these ab.
I only have a small amount of lysate/sample so coating it is not an option. So I believe the cature/ sandwich ELISA is the best.
I have tried coating the Nunc maxisporp plates (at a couple of conc) with rabbit anti-mouse ENAC or rabbit anti-mouse ATPase. Then add my antigen (mouse lysate) followed by alkalinephatase goat anti-rabbit IgG. I don't get any color.
I have also tried biotinynating the anti-ENAC & anti- ATPAse & then incubating with on Pierce coated strep. plates followed by blocking. Thinking that the would get around what I assume is the anti-enac or anti-atpase not sticking. Then figuring I'd just start my real asy from there.
Add the mouse lysate samples. Detect with the goat anti-rabbit. Every thing is high except for the ones without the biotinalyted AB initially added. this would suggest the 2 is just binding to the 1 AB.
Any useful thoughts or suggestions would be appreciated.
Thanks
Quite correct, you're just detecting the primary Ab. You need to detect with anti-mouse ENAC or ATPase. Best option is to coat with a monoclonal, then for a secondary use a polyclonal against the protein of interest. You then detect the whole thing with HRP- or AP-conjugate against the secondary. For example, capture with the rabbit anti-mouse ENAC (preferably MAb), detect bound protein with rabbit anti-mouse ENAC (polyclonal) ,then add goat anti-rabbit IgG conjugate. This means you don't have to conjugate to your target-specific Ab.