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What protein should I use to block a transfer membrane for a western blot? My name is Srikanth, Iamp;#39;m a product scientist at Cell Signaling Technology, and this is CST Tech Tips. (music chiming) In regards to western blotting, a common question we always get is, what should I use, milk or BSA to block? So the purpose of blocking step is to reduce the amount of background due to non-specific bonding. Now BSA is only made up of one protein, BSA, at 60 kDa, whereas milk is made up of many proteins, all of various sizes. So you get a much better chance to reduce more of the background banding. We recommend that you use 5% milk in TBST, shaken for one hour at room temperature, to block all of our non-conjugate primary antibodies. Now this includes phospho-specific and total antibodies. Now I can already hear the clicking, comments, and hashtags, asking about what about the phosphatases in milk? Well, there are some papers out there that discourage you from using milk for phospho signa
