Photo courtesy of Ella Mullins
Milk – it does the body, and the Western, good – usually.
Milk is probably the most commonly used blocking agent in the lab. And why shouldn’t it be? It’s highly soluble and easy to make, it’s inexpensive, and if you run out, you can just pick some up at the local grocery store. It’s also a pretty good blocker; it has a variety of proteins that bind to unoccupied sites on membranes preventing non-specific binding of antibodies and detection reagents, therefore decreasing background noise.
But sometimes milk is not the answer for blocking your Western blot
Milk contains large amounts of casein, a family of related phosphoproteins. In fact, casein comprises 80% of the proteins found in cow’s milk. While casein is responsible for cheesy goodness, it can wreak havoc if you are detecting phosphoproteins. The highly abundant casein proteins will react with anti-phospho antibodies leading to high background.
Milk is also a problem if you are using biotin-avidin detection systems. While this detection method can amplify your signal and help detect less abundant proteins, it is incompatible with milk blockers because milk also contains biotin.
A note on the use of milk: Many protocols use 5% milk as a blocking solution; however a high concentration of milk can actually mask some antigens, making them harder to detect. Therefore if you are using milk, we recommend starting with 1% milk.
How do I go dairy free?
If you do find that your Western is milk-intolerant, there are several other products you can use. A good alternative for a blocking reagent is bovine serum albumin (BSA; Fraction V). Like milk, BSA is frequently used and is a good choice when working with phospho antibodies. Alternatively, you can use single protein blockers. For example, isolated casein protein can be used to block Western blots and avoid interference with other components in milk, such as biotin.
Dairy and protein free?
If protein blockers aren’t working for you, you can always try a protein-free blocker. Protein-free blockers can avoid cross-reactivity seen with protein blockers and also reduce interference with downstream detection. AdvanBlock-PF is a protein-free blocking solution, optimized for use with the WesternBright MCF fluorescent Western blotting kit and is also an excellent blocking reagent for chemiluminescent Western blotting.
So even though milk may be your go-to blocker, remember, you have other dairy-free options!