If the blocking period's purpose is to prevent non-specific binding of the primary antibody in the following step, why do many protocols use the detergent TWEEN in the blocking buffer? Don't we want non specific binding of the blocking protein (caseins or BSA) during the blocking incubation?
Low concentrations of TWEEN in the blocking buffer (and often in the buffer for 1st and 2nd antibodies) are used to prevent non-specific protein-protein interactions. The specific binding is usually more resistant (strong) to this detergent. But non-specifically bound proteins (week interactions) can be washed out (or prevented from binding). Casein and BSA are used to imitate non-specific protein-protein interactions and replace all potential non-specifically bound proteins that might be immunopositive for 1st or 2nd antibodies or just show high non-specific background. The combined use of TWEEN and Casein (BSA) provide the best conditions to decrease most of the non-specific binding.
the main purpose of tween in the blocking buffer and subsequent steps is to prevent none specific binding of proteins. I have attached a file below that provides a nice explanation.
http://csmedia2.corning.com/LifeSciences/media/pdf/elisa3.pdf
Low concentrations of TWEEN in the blocking buffer (and often in the buffer for 1st and 2nd antibodies) are used to prevent non-specific protein-protein interactions. The specific binding is usually more resistant (strong) to this detergent. But non-specifically bound proteins (week interactions) can be washed out (or prevented from binding). Casein and BSA are used to imitate non-specific protein-protein interactions and replace all potential non-specifically bound proteins that might be immunopositive for 1st or 2nd antibodies or just show high non-specific background. The combined use of TWEEN and Casein (BSA) provide the best conditions to decrease most of the non-specific binding.
So, isolating my question to only the blocking step of a Western blot (before any antibodies are introduced to the membrane), would you say that TWEEN should be in the blocking buffer to prevent the milk proteins from binding to each other..? and why would that matter, as they would be washed out anyway?
TWEEN should be in the blocking buffer to saturate all potential non-specific binding sites on your membrane. When all of them blocked by TWEEN the non-specific proteins in your 1st antibody solution (and milk) will not be able to bind to the membrane. In our lab we usually wash the membrane with water after the transfer, let it dry on the open air, and then incubate directly with the 1st antibody solution containing TWEEN and 5% non-fat milk. Even when the 1st antibody is mouse antiserum we never see any problems with high background or non-specific binding.
Detergents (tween 20) are considered temporary blockers; they do not provide a permanent barrier to biomolecule attachment to the surface because their blocking ability can be removed by washing with water or aqueous buffer. To be useful as the sole blocking reagent in an assay, detergents must be present in all the diluents/buffers subsequent to coating the surface with a capture molecule. However; when used in conjunction with a protein blocker, detergents provide added convenient and inexpensive blocking ability during wash steps, etc. by blocking areas on the surface that may become exposed due to protein/biomolecule desorption.
I think she is asking why you should add tween in the blocking step, before adding any antibody... of course it make sense to add it while the primary or secondary antibody... but I still don¡t get why is added in the blocking step
This is a great discussion. I am working on developing and testing a new type of optical biosensor. Thus, trying to learn more and modify common ELISA protocols. In terms of Twee 20 - would it be advised or dis-advised to use it for diluting patient plasma/serum?
Thank you!
Alex.
My guess is that the Tween is added to the blotto buffer for the incubation and washing steps. As milk powder is dissolved in blotto there is also tween present in the blocking step, where it isn't strictly required. People (including myself) are simply too lazy to make two separate batches of buffer, with and w/o detergent. And that's ok, since that little detergent doesn't do any harm during blocking.
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