The options that you mention are just options.
Both blocking and sample dilutions can be prepared with different blocking agents like BSA, milk, or Tween 20. Serum can also be used, but you have to be careful. If you are going to use an anti-goat conjugate, you cannot block the plate with goat serum, for instance.
If you dont add any blocking agent to your antibodies and/or conjugates, it can be a high non-specific background.
You should try several blocking/diluting solutions to achieve the best signal/noise ratio. If you already have a protocolo that Works, you can use it, of course, but probably this is not the only possibility.
The Tween blocks non-specific binding by reacting to the styrene residues, preventing proteins from reacting via their tyrosine residues. 5% is high enough of a concentration to achieve saturation. Typically, 1-5% BSA will do the same thing.
I would need to know what your detection antibody is to be sure, but it sounds like the normal goat serum is added as a source of goat IgG, which I am guessing blocks cross reaction of the detection antibody with other antibodies. We often used either normal rat serum or normal mouse serum to block cross reactions in secondary fluorescent antibody staining protocols.
5% PBST is not used for Blocking generally. However, a Max Concentration of 5% Skimmed Milk Powder dissolved in PBS buffer with 0.05% Tween 20 is commonly used for blocking and it works simply great.
I concur that 5% Tween is not commonly used, but it does work. Likewise, 1-5% BSA, casein or powdered milk can be used, but there is a hook with using powdered milk. The powdered milk does contain dried bacteria as well, and the bacteria can interfere with binding of specific antibodies, most notably anti-phosphoryl choline antibodies. We had that problem in the lab I worked in out in San Diego. For the anti-PC plates, blocking and washing was done with BSA containing solutions only.
@Gary, you are correct in the quality of milk powder. It's the least coated option for blocking the plates. If anyone is looking for high specific results, they have to scrape off the Milk Powder from the menu and use BSA instead.
Every lab (and sometimes even every ELISA) has a preferred blocking agent. In my lab 4% milk is successfully used for almost all our experiments (with better results in general as compared to BSA). I had to switch to BSA only once for a particular experiment, because the anlyte was present in milk.
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