In western blot why do we dilute primary antibody in 1% skimmed milk so that if there are any antibodies against blocking agents they get blocked? but why would they be antibodies against skimmed milk?
and 1% BSA for secondary antibody dilution, Goat anti-Rat HRP antibody why do we use BSA here?
Hello Xin, in our lab we in fact use 1% BSA for both the initial blocking step, the primary and the secondary antibody dilutions - so you can use the same blocking agent in all cases. In principle, you need a relatively cheap protein that 'covers' plastic and non-specific sites on the membrane where the antibody could otherwise bind to.
Well, because the non-specifically bound proteins tend to dissociate fast, meaning that if you leave your membrane long enough in buffer only, the blocking protein comes off the membrane. So you make sure your antibody either stays in solution or binds specifically to the antigen on the membrane.
Blotto, the buffer used for western blotting, is in effect PBS with tween and a carrier protein. Both Tween (a non-ionic detergent) and carrier protein reduce background by unspecific binding of antibody to the membrane.
BSA can be used as carrier protein, but its homogeneous nature makes it less effective than, for example, milk protein. The exception are blots against phosphorylated proteins, as milk contains phospho-proteins.
The first incubation of the membrane is in a solution of high protein concentration, say, 5% non-fat milk powder. Recall that you have used a high protein-binding membrane to catch all proteins that move out of the gel onto the membrane . If you would incubate directly with your first antibody, all your antibody would be bound all over the membrane, not to the target protein. This would result in a totally black blot. You first need to saturate the protein binding centres on your PVDF or NC membrane.
I understood the purpose of blocking as Darja Lavogina said non-specifically bound proteins tend to dissociate fast which raised a doubt eg if I use 5% skimmed milk for blocking during wash with PBST for 5min 3 times on rocker some blocked and loosely bound proteins could strip of the membrane? and primary antibody dilution in 1%SM PBST and secondary in 1%BSA PBST could alleviate the background issue?
Nice point, BSA is homogeneous while skimmed milk is not but would that really matter all this time I was under the impression skimmed milk is cheaper than BSA so 5% SM is used for blocking?
You need to distinguish between the initial blocking step (say, with 5% milk powder) and carrier protein (say, 0.1% milk powder) in all further steps. The former blocks the membrane, the latter prevents unspecific aggregation and absorbtion of low concentration protein (e.g., antibodies) to hydrophobic surfaces. It has little to do with the speed of association.
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