Milk or BSA? Choosing a blocking protein for Western Blotting (WB) | CST Tech Tips

What protein should I use
to block a transfer membrane for a western blot? My name is Srikanth,
I’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 signal. Let me address that by saying that in all of our in-house testing, we don’t see any of these issues. CST scientists run so many westerns that we end up making milk
at least once a day, sometimes multiple times a day. Now if your milk buffer goes unused for a week, two weeks, even longer, that increases your chances of phosphatases affecting your signal. But again, we don’t see
any of these effects, because we use milk buffer
fresh on a daily basis. These are images of product #13038, tested on lysates made from
3T3 cells treated with PDGF. The only difference between the blots is that one membrane is blocked in milk, and the other membrane is blocked in BSA. Clearly, the membrane blocked in BSA has a much higher background. Yet phospho signal is
still strong and clean with the membrane blocked in milk. So after the membrane has been blocked, please refer to the
product’s specific data sheet for the recommended
antibody dilution buffer. It’s either gonna be milk or BSA, depending on the specific antibody. I hope this has been helpful. For full application specific protocols, they’re available on on the specific product page. If you have any other questions, please feel free to contact
any of the scientists at CST at For more CST Tech Tip videos, please subscribe to our YouTube page. Good luck with your experiments. And we’ll see you next time, thanks.

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