Why milk is used in Western blotting? - (Oct/05/2006 )

hi all..

through my readings , i notice that milk is widely used for WB..what exactly makes milk useful here and why some protocols make use of nonfat-milk?!



thanx in advance...

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Hi,

Milk contains a lot of proteins which bind to the membrane to reduce background, non specific binding. Antibodies are proteins, so they can bind to the membrane inespecifically, this is, they bind to any site in the membrane (only due to being proteins) apart from binding to their antigen.

So, after transfer, proteins contained in the milk bind to the membrane and, thus, fill a a lot of sites ("potential background sites") there. After this, when you incubate with your antibody, it binds to the antigen (the "perfect" situation is when your antibody only binds only to your antigen and not any other site in the membrane) and has less possibilities of inespecific binding.

Some people instead of milk use a BSA solution and there are available some commercial background reducing powders, but for me milk has always worked fine. Furthermore, I dilute my secondary Ab in 3% dry milk in TBS 1x and this also has helped me to reduce background.

Well, I hope I´ve been clear, if not, I´ll try to explain it again. Sorry for my english.

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there are situations where milk is not appropriate. milk contains phosphoproteins (eg-casein) and biotin. if you are working with anti-phosphoprotein or with biotinylated antibodies then your backgrounds may be too high.

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I think the main reason to use milk protein was still not answered: It is the low immunogeneity of its proteins which should reduce unspecific background in Wb; the same is true for serum albumine; reduction of fat enhances binding of proteins not disturbed by triglycerides

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and I would say, at least in our lab, the reason milk is preferred where possible is because it is so much cheaper than BSA. BSA would be the ideal for us in all applications, but it's very expensive compared to milk, which works for most applications

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pumuki, mdfenko, kosmodrom, and aimikins

thanx alot for your replies

what i understand from your posts is that milk is used to prevent non-specific binding of antibodies to the membrane...right?

causes:
- its proteins bind to the membrane thus not giving antibodies the opportunity for non-specific binding
- immunity: milk proteins have low immunogenity

correct me if i am wrong...

mdfenko: can u explain your post more, PLZ

and one more basic wondering that i don't know: what do u mean by "membrane background" ?

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Membrane background are signals caused by inespecific bindings of the antibodies, when they bind other sites in the membrane different from the antigen. This difficults the analysis, because the band is not clear.
I´m a beginner in WB, but if I can help you more, here I am.

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sure, first, let me retract the statement about biotin. i had been told, years ago, that there was significant amounts of biotin in milk but i found that is not the case (unless the milk is supplemented). so disregard that part of my post.

however, milk does contain phosphoproteins, more specifically, casein. casein is a major protein component of milk and will bind to the membrane when blocking (this is what you want to happen). a problem arises if you want to probe your blot for a phosphoprotein with an antibody specific for the phosphorylated site on the protein (usually a specific phosphoamino acid). many of these antibodies will bind to the casein as well and will give rise to high background on the blot. in those cases you would need to switch to a more neutral (in the sense that it won't react with or be recognized by your antibodies and/or substrates and/or reagents) blocking agent (eg-bsa or some of the non-proteinaceous blocking agents).

hope this explanation is clear. don't hesitate to ask more if it isn't.

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once again, thanx pumuki and mdfenko very much

it's more clear now

to be sure:

"blocking"= covering the membrane to avoid non-specific binding...right?
and "dark background" means too much non-specific binding!?

mdfenko: u mean that casein is a phosphorus protein, right?

what other blocking agents one could use (proteinaceous and non-proteinaceous)?

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As Aimikins said, other possibility is using a BSA solution, and also some brands have available blocking "secret" solutions or powders.

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