I always use 4% milk/PBS during panning and screning. Milk is ideally suited to block non-specific interactions in many experimental systems. I also have used BSA and in most cases it also works well, but in a few cases milk was clearly better than BSA to avoid non-specific interactions. Thats why I routinely use milk.

Sometimes panning result in enrichment of sticky non-specific phages. Succesful specific selection can usually overcome this enrichment. But if selection was not good, you can have at the end mostly theses non-specific binders. Using BSA as blocking agent, these properties can be more evident. When you switch to milk, reduction of non-specific background can result in total loss of binding, reflecting that there is no background and no specific selection. In that case, you would have to rethink your panning strategy.

I assume there is no reason to expect specific interference of milk/BSA in the interaction you are looking for, and that when you talk about background without coating, you blocked the wells with BSA/milk before going on with the ELISA.

Hi Gertrudis,

Thanks for your responds, and I understand I didn't get specific binding after selection, that's the reason I get strong signal when I use BSA block even no antigen coated. But I still don't understand why the signal gone when I use milk block. it's just the milk is better for blocking non-specific binding?

Yes. Casein is very small and very, very hydrophobic. So it blocks every single aggregation-prone site on both your surface and your soluble molecules ore efficintly t.han BSA In most cases BSA is as good as casein, but in some cases background is oly nremoved wit casein. As casein is extremely dficult to dissolve , many people prefers to use casein-rich milk instead.

Hi Gertrudis, I appreciate for your reply , it help me a lot!