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.

---

other Qn:

- 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.

---