Elisa

[Guest michael789]

Does anyone know of any good ELISA blocking agents? Im doing a two step ELISA where i bind the first protein then block then bind my secondary protein but i cant stop my secondary protein from binding even if there is no primary protein and ive blocked the well ( or attempted to)

Ive used BSA, skim milk, peptone tryptone, egg yolk.

Thanks

[daisy]

Looks like you've tried what I would have tried but maybe you could try a different serum such as rabbit, goat, donkey etc. You could also try just blocking with tween-20 (0.1%). Good luck - I know your frustration!

[blike]

serum such as rabbit, goat, donkey etc

 

These are the actual names of them??

[daisy]

Hehe. Well, it just means blood serum from donkey, rabbit, goat - whatever. Polyclonal antibodies are often raised in rabbits, sheep etc. so it's common practice to "block" non-specific binding by incubating tissues, cells etc. with the serum your secondary antibody was raised in.

[blike]

Haha, gotcha.

[daisy]

Very funny Blike - and here I was thinking I was sharing pearls of wisdom:embarass: !!

[daisy]

Actually Michael789, try a google search for an immuno forum site and maybe you can get a definitive answer from people who've being doing this longer than me. I'm still relatively new to immuno.

[Guest michael789]

thanks everyone for your input

If you know of anymore good forums or sites or anything please post it.

Help is always appreciated

[daisy]

Try this site http://micro.nwfsc.noaa.gov/forums/. There are some good Q&A pages on this. Also there is another site dedicated to immuno/in-situ etc. but I've got it bookmarked elsewhere. But actually I think it's mentioned on the other sites immuno forum anyway. Good luck.