beginner's Questions on Western Blotting - (Jun/14/2009 )

Hi. I am new to western blotting.

I would like to know, why non-fat milk, but not whole milk, is used in blocking?

Another question is, why do i need to add the antibody one by one (add primary antibody, incubate, rinse, then add secondary antibody)? What's the reason behind? Can i mix the primary and secondary antibody first, incubate for a period of time, and then add to the blot?

Thanks!

Why non fat milk? fat and water dont mix and you need the milkprotein to block.
Why not mix the primairy and secondairy in one step?
Because you will get the best results as the binding places at the primairy will be fully availlable.
For a great number of antibody's you can buy it with allready coupled detecting group.
For the mixing of the antibodys you can try it and i assume it will work most of the time but I also think you will get a lost of sensivity,

Gerard is mostly right:
The reason you add the primary and secondary separately is that the secondary will cause steric hindrance of the primary binding to the specific epitope. Another reason is that if you add the secondary at the same time you increase your background as the secondary may bind to the membrane non-specifically, whereas if there is a primary there, the secondary is much more likely (thermodynamically) to bind to it.

also, let's not forget that antibodies will precipitate with antigen (ip anyone?). so, in this case, most of the primary antibody will be taken out of the equation.

I knew there was another reason, but couldn't for the life of me think of it.