Chemilumenescent EMSA

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Ms NO's picture
Chemilumenescent EMSA

I need a good blocking buffer for my chemiluminescent EMSA. The kit I started with will not tell me what is uses in the blocking buffer. I no longer need the kit except for the blocking buffer, and $300.00 per 500 mls is alot to pay. I am using biotinylated target, running it out on a native 7.5% Acrylamide gel and transfering it to a positively charged nylon membrane and then using a SA-HRP conjugate, and developing with a chemiluminescent substrate. All is working well, but I need a good blocking buffer.

samm's picture
The only suggestion (which i

The only suggestion (which i'm sure you've received by now) would be to try out different combinations of DNA and protein spp blocking agents (e.g. salmon sperm DNA+fish skin gelatin OR milk powder OR BSA) across multiple strips of your nylon membrane (each with one lane loading with exactly the same amount of your positive control).