Hi there,

we try to establish a multiplex western blotting method using fluorescence-conjugated sec. antibodies (anti-rabbit IgG StarBright Blue 520 and anti-mouse IgG StarBright Blue 700 from BioRad). As BioRad recommended using casein-TBS-buffer (1%) for blocking, I ordered solid casein and we tried to dissolve it in our TBS-buffer we normally use for other western-blotting methods (TBS buffer containing 10 mM Tris HCl and 150 mM NaCl, pH 8.0). The problem was, that we weren't able to dissolve the casein properly, even when we ajusted the pH again to pH 8.0. Is the only thing we could do ordering the one from BioRad (ready prepared) or has anyone an idea or a recipe? Usually, we use non-fat dry milk (5%) for blocking in TBS-T which works pretty nice for most of our blots. As a result, we had a very hight background with the casein-TBS-buffer which suprised me, because BioRad recommended using casein instead of milk as it would decrease high background. Could it be, that we had the high backgorund because of the precipitants of casein in the blocking-buffer? We used low-fluorescence membranes, so that should not be an issue.

Thank you so much for you help!

Bests, Charlotte