TMB Substrate Creation

July 6, 2006 6:52 PM

Biology Research Filter: How do you make your own TMB substrate solution for use with an ELISA kit?

My research group is running immunassays for used detection of aldosterone. We're tired of buying the plates ($250 a pop), so we're looking at producing our own. The TMB substrate for use with the HRP-conjugate is part of the reaction that is included with all of the purchased plates, but we're having trouble creating our own. The companies don't give out any concentrations or directions for creating solutions, since they want you to purchase their own. We've found several protocols that involve a citric acid buffered solution, but our TMB won't dissolve. It will dissolve in DMSO, but our results aren't great. I understand that this is a little specific, but I'm sure someone out there in the vast world of MetaFi has experience with this. Thanks for the help.

posted by sicem07 to science & nature (4 comments total)

You might try first dissolving in ethanol and then into your buffer. I imagine that ethanol, which will dissolve TMB, would be less inhibitory than the same volume of DMSO. Pre-made and stabilized TMB solutions are also commercially available on their own.
posted by shoos at 7:43 PM on July 6, 2006

Pierce sells a TMB substrate solution. Have you tried that? I've had very good luck with Pierce products.
posted by Quietgal at 7:46 PM on July 6, 2006

Can't you buy just the substrate solution by itself from somewhere? It will probably still be cheaper than buying the kits. It seems Sigma sells it.
Otherwise, I don't know, I've never done ELISA, but I think one of my coworkers might have done it in the past, so I can ask her. But she's a big fan of kits so I doubt that she'd know how to make her own TMB solutions.
posted by easternblot at 7:47 PM on July 6, 2006

I second the Pierce reagent. Our lab has had good experiences with Pierce. Good Luck!
posted by LunaticFringe at 5:38 AM on July 7, 2006