Western Blocking Reagent, Solution

Western Blocking Reagent is used as a general blocking agent during western blotting experiments and immunofluorescence staining. The reagent is used in steps such as blocking the membrane, membrane washing steps, and diluting detection antibodies.

Each lot is function tested (dot blot).

The Western Blocking Reagent is a 10x solution that contains 10% purified casein protein in maleic-acid buffer.

Working solution: PREPARATION OF WORKING SOLUTIONS

• Tris buffered saline (TBS), pH 7.5

To 800 ml redist. water add 6.05 g Tris base (50 mM), 8.76 g sodium chloride (150 mM) and adjust the pH to 7.5 with approx. 9.5 ml 1 M hydrochloric acid. Then dilute to 1 l with double-dist. water. TBS is stable for 3 months, when stored at 2 to 8 °C.
Note: Since sodium azide inhibits POD, it must not be used as antimicrobial agent when using POD-conjugates.

• TBS-Tween (TBST)

Washing buffer 1: Dissolve 1 ml Tween 20 in 1 l of TBS. TBST is stable for 3 months, when stored at 2 to 8 °C.
Note: 0.1% Tween 20 is suitable for the most applications, but—depending on the membrane and on the antibody used—different detergents (like SDS, Triton X-100 and Nonidet P40 and detergent concentrations from 0.01 to 1% may lead to better results.

• Blocking solution (1%)

Dilute 10 ml Western Blocking Reagent (10x conc.) in 90 ml TBS. The Blocking solution can be stored at 2 to 8 °C for 1 month.
Note: Since sodium azide inhibits POD, it must not be used as antimicrobial agent when using POD-conjugates.

• Blocking solution (0.5%)

Dilute 50 ml Blocking solution (1%) with 50 ml of TBS.

• Antibody solutions

Dilution and incubation solution for all antibodies is 0.5% Blocking reagent in TBS (see above). In order to exploit the full detection potential of the system we recommend to optimize the dilutions of the primary and secondary antibody in dot blot assays in advance. (Start first with 3 to 4 dilutions of primary antibody and a constant concentration of the second antibody. Then choose the most suitable dilution of primary antibody and optimize the concentration of the secondary antibody in the same way.)
Note: The concentration of the blocking reagent is an important parameter for improvement of the signal to noise ratio in Western blots. If high background appears even under optimized antibody concentrations, increase the concentration of the blocking reagent during the antibody incubations and washing steps from 0.5% to 1%. In case of weak signals even with prolonged antibody incubations lower the concentration of blocking reagent during the antibody incubations and washing steps from 0.5% to 0.1%.

For life science research only. Not for use in diagnostic procedures.