Why do we need BSA as a blocking buffer for certain antibodies? What is different from casein?
Thanks,
Anand
BSA and casein are used to block non-specific protein binding. Casein is cheaper than BSA and is suitable for most applications, However, I do know of at least one application were casein is NOT suitable as a blocking agent, and that is for detecting anti-phosphoryl choline antibodies in ELISA. The reason for this is that casein is derived from milk - most typically spoiled or out-of-date milk - and has significant amounts of bacterial debris present, which includes many different bacterial antigens, notably teichoic acids, which often contain phosphoryl choline. My guess is that whatever antibody you are using for whatever application, it cross-reacts with some bacterial antigens found in casein, thereby obstructing your assay.
BSA and casein are used to block non-specific protein binding. Casein is cheaper than BSA and is suitable for most applications, However, I do know of at least one application were casein is NOT suitable as a blocking agent, and that is for detecting anti-phosphoryl choline antibodies in ELISA. The reason for this is that casein is derived from milk - most typically spoiled or out-of-date milk - and has significant amounts of bacterial debris present, which includes many different bacterial antigens, notably teichoic acids, which often contain phosphoryl choline. My guess is that whatever antibody you are using for whatever application, it cross-reacts with some bacterial antigens found in casein, thereby obstructing your assay.
Your guess may be correct, as I see most of the anti phospho antibodies are suggested to use with BSA. thanks for your comment :)
You are quite welcome. When I was a post-doc, I was working in a lab that did hundreds of ELISAs a week, and there was a need for high-throughput platewashing. We bought a system that was basically a block with 96 holes fitted with 200 microliter tips that sprayed a jet of wash solution upwards. It was fed from a 20 liter carboy. The cost of using BSA in the wash solution was prohibitively expensive, so I tested casein as a substitute. It worked well for our assays, but when another PI used it for her anti-phosphoryl choline antibody ELISA, the assays did not work. We tracked it to the casein. So she had to block and wash her plates with BSA solutions by hand. Fortunately, she did small assays - 5 plates maximum, so it wasn't a hardship.
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