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

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!

serum such as rabbit, goat, donkey etc

 

These are the actual names of them??

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.

Haha, gotcha.

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

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.

thanks everyone for your input

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

Help is always appreciated

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.