Why milk is used in Western blotting? - (Oct/05/2006 )
Just wondering what if the milk blocks the antigen that we are interested in?
In my opinion, this is very improbable.
Milk proteins bind the membrane because the membrane has high affinity for them as well as for any protein. But the antigen is a specific protein with a specific structure who only should bind its antibody, so I think it´s very improbable that any other protein different from the antibody can bind the antigen.
may theoretically happen if milk protein directly bind to antigen and mask epitop if you use a monoclonal; for a polyclonal it may no problem as not all epitopes should be blocked; nevertheless, I think milk-blocked antigen it´s a rare phenomenon
yes, casein is phosphorylated. it contains phosphate bound to certain amino acids.
one more Qn: how milk is applied in WB, i mean how to prepare it?
Usually, a 3%(w/v) buffer solution. I've seen many ways of incubating and blocking nitrocellulose membrane. The most common procedure is probably in a petri-dish on a shaker with ~30-40rpm for one hour. Thereby, the membrane floats in the dry milk solution. For AB incubation many scientists only add there AB later to this solution (keeping dilution in mind).
I prepare it at 5% in TBS1x.
- why "non-fat milk" is used? what's wrong with whole fat milk?
is there a relation with binding to the membrane?
- what is the role of tween in WB??
I don´t know exatly but I suposse that fat milk gets "dirty" the membrane, I think that difficults the binding of the antigen with the antibody.
Tween is a detergent (well, a registered trademark I guess) and it´s used in the wash buffer.
On TBST works to me.