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I am currently learning protein identification techniques and come across ELISA and Western Blot. In these methods, a washing buffer is required to wash out the antibodies that are not bound to the plate (the specific antigens).

Questions:

  1. I understand that detergent (E.g. Tween-20 (T)) is usually added since it can wash away the non-specific binding of antibodies. (ref) However, from what I have known, we are also adding blocking buffer to block the unoccupied sites on the membrane, which also prevents non-specific binding of antibodies (ref). I would like to ask if this is true, why do we need to add Tween-20 to the washing buffer?

  2. I have heard PBST and TBST are 2 common washing buffers, where TBST seems to be much better. From ThermoFisher:

Determining the proper blocking buffer can help to increase the system’s signal-to-noise ratio. Occasionally, when switching from one substrate to another, the blocking buffer may need to be changed in order to avoid problems with diminished signal or increased background. For example, with applications using an alkaline phosphatase (AP) conjugate, a blocking buffer in Tris-buffered saline (TBS) should be selected because phosphate-buffered saline (PBS) interferes with AP activity.

This seems to me that TBST is always the preferred option (also ref). Then I would like to ask in what specific situation, that PBST is preferred than TBST, otherwise, it will cause a major problem to the result e.g. signal/noise ratio?

Thank you.

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  1. All the unoccupied sites on the membrane are saturated in the blocking step after the transfer of the protein gel to the membrane. You can prepare the antibody solution in pure buffer, but you should expect a slightly higher background (this of course depends also on the antibody used), since there still may be some binding possible. When you have a much higher amount of blocking agent in this solution it is much more unlikely that the antibody is able to bind non-specifically, since these sites are occupied already.

    Some low affinity binding occurs between the antibody and other proteins which you will not be able to wash away without a detergent.

  2. In my experience it doesn't really matter for most cases which buffer you use as long as you do it consistently. There are of course a few exceptions:

    • When working with phospho-proteins PBS can give a much higher background - but it can also work. It needs to be tested and optimized, but it is probably easier with TBS.
    • When using alkaline phosphatase in your detection step, you cannot use PBS since free phosphate inhibits the enzyme.
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