My protocol is to block my sample with BSA 1% in PBS-T for an hour at room temp and then add antibodies diluted in BSA 1% in PBS-T. Why do I need to dilute my antibodies in BSA if I do the blocking step or vice versa why do I need to do a blocking step if I dilute with BSA solution?
Follow the protocol. Presence of blocking buffer will further minimize the nonspecific interaction of antibody and enhance specific signal of your protein of interest.
Best
Hello Jillian Porter
Blocking is an important step as it prevents antibody from binding non-specifically. Diluting antibody in blocking buffer (BSA 1% in PBS-T) can avoid the unpredictable non-specific binding, thereby the antibody will bind to the interested antigens more specifically. If you have performed the blocking step for 1 hour in 1% BSA in PBS-T, the choice of diluting the antibody either in PBST/TBST or blocking buffer will depend upon the antibody.
The ultimate goal is to reduce cross-reactivity and minimize non-specific binding. Since antibody preparation vary in their level of purity and specific binding properties, there will be differences when using antibodies for various assays like ELISA, Western Blot, etc.
I would recommend even after blocking in 1% BSA in PBS-T for 1 hour, you should dilute the antibody in 1%BSA in PBS-T to obtain clean and good result without much background signal.
Best Wishes.
BSA in blocking buffer binds to the surface where proteins are not bound (or blocks the empty space on the blot), it won't allow primary antibody to bind to empty space on the blot. Blocking is essential. Diluting antibody in blocking buffer helps in reducing non-specific bands.
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