Hi all, sorry to be peppering this forum with questions.
My colleague and I were wondering what problems might happen from buffers that have an improper pH, and how close to the desired pH one must be before those problems don't happen.
For instance, he was a full point off(!) when he created his TBS10x buffer, and by extension, at least a full point off when he did TBS-T1x. The reason is, is that our water is highly basic. The desired pH was 7.6 and he was at 8.6. He got no signal when he ran his western blots. I was theorizing that perhaps the additional negative charge my alter the shape of the proteins, inhibiting the binding of the antibody to the active site. That would mean that monoclonal antibodies would be different from polyclonal, so maybe that answer is wrong.
Anyways, how close should you be with buffers? 0.1, 0.01? I made it and I'm .04 off - more acidic, I think that's acceptable though as I'll be diluting it with that basic water.
Do you know what mechanism the pH of buffers affects the western blot process, and how close we must be with them?