I was incubating my tissue with primary antibodies in a solution of PBS, DMSO, Tween20 and 3% milk at 37°C. Unfortunately, the milk started to curdle. Will this affect my experiment drastically? Are there any alternatives I can use? Like BSA instead? Any help would be very much appreciated!
Hello Hridya Harikumar
Yes, curdling of milk would affect the binding of primary antibodies. The effect of pH on the equilibrium constant of the antigen-antibody complex lies in the pH range of 6.5 and 8.4. Below pH 6.5 and above pH 8.4, the antigen-antibody reaction is strongly inhibited. Curdling of milk will make the primary antibody solution acidic as it will bring down the pH in the range of 4.5-5.5.
As an alternative, you may use primary antibody dilution buffer namely, (1X TBS, 0.1% Tween-20 with 3% BSA).
Best.
