Need help with Pierce - (Apr/24/2002 )

Hello all,

I am trying to figure out what protein is used in Pierce's Superblocker.  I am doing this because our lab likes to use it for our FLAG based purifications and then HRP detection on western blots using ECL Plus.  It blocked absolutely great for that (not too much not too little), but recently we tried out Pierce's Superblocker on a HIS-tagged expressed protein, and it blocked all signal from the HRP and even gave a strange bown tint to the membrane when ECL Plus was applied.  We have gotten other blocking solutions to work with this HIS tagged protein detection, but I am stumped to why our lab favorite Pierce's super blocker wont work.  I am assuming that it is the protein they use in their blocking solution that is causing the total blockage of all signal, since the amount of buffer between that and the other blockling solutions I tried (milk, casein) is the same.  Any ideas, suggestions.  We really liked the Superblocker (great signal to background ratio) but if we have to give it up for HIS-tagged detection, I suppose we have to.

Robert,
As you know, there are many keys in which to play the blues. Why worry about this blocking agent? Peirce makes another great kit for His tag detection - India West Pico (something like this). The blocking is done with TBST/BSA and the detection is done with Ni++ activated HRP - no antibodies. I've seen localized brown tint on my westerns before with very stong reactions using ECL substrates.

Robert

We had this problem with Superblocker. It has nothing to do with your His Tagged protein and everything to do with proteins getting inactivated or denatured in the superblocker. Store your superblocker at 4 C AND warm it at 37C for 1 h prior to adding it to the blot.

Good Luck

Sam Brilliant
dstf@doctor.com
Ph.D. Rescue Team
A division of DSTF-Global
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