So what is fluorescence SUPPOSED to look like? - fluorescent secondary antibody scans as SOLID BLACK image (Nov/20/2008 )

Hullo all,

Thanks for stopping by -

My PI and I would like to use a fluorescent 2' antibody to visualize some DNA on a membrane. When I scan the chemiluminescent-treated control, it shows a medium/light background with dark dots, as I would expect. When I scan the fluorescent-treated membrane, it results in a solid black image. Like, the whole membrane is black. It was scanned under the parameters provided with the protocol, and the membrane is compatible with fluorescence. I tried not to expose it to too much light, but I don't know how good I was at that.

Is this a case of everything fluorescing (i.e. I need to dilute my antibodies waaaaaaay more.... I'm getting the same response from 1:2500 and 1:5000 dilutions) or NOTHING fluorescing?

Should I play around with dilutions, or try washing it more? Did I expose it to too much light?

We don't block these membranes.

Here is the protocol:

Amersham ECL Plex Western Blotting System
1. primary antibody for 1.5 hours at room temp, shaking
2. Rinse membrane in TBS-Tween, wash 2X in TBS-Tween for five minutes each, shaking at room temperature
3. Add 2' antibody (recommended dilution 1:2500) and shake for one hour, protected from light.
4. Rinse membrane in TBS-Tween, wash 3X in TBS-Tween for five minutes each, shaking at rt, protected from light.
5. Rinse membrane three times in TBS.
6. Scan using fluorescent laser scanner on setting "580 BP Cy3 TAMRA" filter


Please help! Thank you so much!

Amersham ECL Plex Western Blotting System is for proteins, and I do not believe it will work for DNA. Maybe a southern blot protocol would suit your needs.