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This protocol has been routinely used for western blot membranes measuring 10 cm x 10 cm, 10 cm x 20 cm and 20 cm x 20 cm. The protocol may have to be optimised by the user to produce the best results.

1.      Prepare and run SDS-PAGE gels of samples (Laemmli, 1970. Nature 227, 680-685) and transfer proteins to PVDF membrane.

2.      Block non-specific binding sites by incubation of PVDF membrane in 200 mls of TBS-Tween/milk (TBS: 50mM Tris-HCl pH 7.4, 150mM NaCl, + 0.1% v/v Tween-20 + 5% w/v non-fat dried milk powder) for 60 minutes at room temperature with continuous, gentle rotary agitation.

3.      Incubate PVDF membrane with primary antibody at a dilution recommended on the product specification sheet (in TBS-Tween/milk) for 3 hours at room temperature, with continuous gentle agitation, or for 16 hours overnight at 4 °C.

4.      Whilst preparing the secondary antibody, wash the PVDF membrane in 200 mls of TBS-Tween with continuous, rotary agitation. Agitation should be vigorous but not too much so as to damage the membrane. If the PVDF membrane was incubated with the primary antibody overnight, additional washes may be required.

5.      Incubate the PVDF membrane with anti-rabbit IgG-peroxidase or anti-mouse IgG-peroxidase secondary antibody as appropriate* (1:5000 dilution (in TBS-Tween) was used in the examples shown on this site) for 90 minutes.

6.      Rinse the PVDF membrane in 100 mls of TBS-Tween twice. Then wash the PVDF membrane in 200 mls of TBS-Tween for 1 x 20 minutes followed by 4 x 5 minutes, with continuous, rotary agitation on each occasion. Again, agitation should be vigorous but not too much so as to damage the membrane.

7.      Use peroxidase detection method of choice (enhanced chemiluminescence was used in experiments displayed on this site).

* Secondary antibody must recognise primary antibody. For example, if the primary antibody was produced in a rabbit, the secondary antibody must be anti-rabbit IgG peroxidase.

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Last modified: December 04, 2006