It is necessary to wash the membrane after blocking and between incubations with the antibodies. Washing removes unbound or aggregated proteins present on the blot as well as unbound reagents that may interfere with the detection of the target molecule(s). Washing helps to reduce background signal, improving assay sensitivity by increasing the signal-to-noise ratio and enabling qualitative and quantitative analysis of the protein of interest. The correct washing reagents should be used at the correct concentrations to ensure removal of deleterious agents from the membrane.
PBS solutions can vary in their formulation, but approximate physiological conditions by combining sodium phosphate and sodium chloride to buffer the solution to a pH of 7.5. If the target protein is phosphorylated, or an Alkaline Phosphatase (AP) conjugate is to be used, PBS should be avoided as the phosphate can interact with phosphorylated proteins or quench the AP activity affecting result reliability.
TBS is sometimes used as a washing buffer as an alternative to PBS. It is typically made by combining Tris Base with Tris HCL and sodium chloride to create a solution with a physiological pH of approximately 7.2.
Detergents such as Triton X-100, SDS, or Tween-20™ are added to prevent aggregation of unbound proteins. Sticky complexes can form and bind to the membrane, causing background Tween-20™ is typically used at 0.05% to 0.1% v/v of the solution.
Concentrations higher than 0.1% can strip the target protein(s) from the membrane and the antibodies bound to them, reducing the signal and accuracy of the blot.
Caution: Microbial Growth can create a background signal
Wash buffers should be made freshly before use.
Caution: Blocking reagents in wash buffers
Blocking reagents are sometimes added to wash buffers. We recommend using only solutions of the buffer, without the addition of blocking proteins like powdered milk or BSA.
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