Western blot

The Western blot or immunoblot is a common technique in cell biology and biochemistry, which permits the detection of a specific protein in a complex mixture of proteins. It was first described in 1979 by two groups led by Stark (Stanford; Proc Natl Acad Sci U S A. 1979 Jul;76(7):3116-20) and Gordon (Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350-4). The term Western blot was introduced by W. Neal Burnette and made reference to the geographical location of the groups involved (West Coast USA) and the existence of DNA and RNA blotting techniques called Southern and Northern blots respectively.

The technique relies upon the separation of protein components by gel electrophoresis (usually SDS-PAGE) and the transfer of proteins to an immobile support made of nitrocellulose, nylon, PVDF or other materials. The proteins immobilised on the membrane support can then be washed free of denaturant (SDS) to facilitate their interaction with a specific monoclonal or polyclonal antibody, or other binding partner (natural protein partner - far-Western blot; RNA aptamer, recombinant antibody or antibody mimetic). The antibody antigen complex is typically detected using a labelled secondary antibody (which detects the primary antibody). Labels are often enzyme molecules (peroxidise, phosphatase) and are often detected using chemiluminescent substrates. Alternative labels attached to the antibodies include fluorophores, radiochemicals, and colloidal gold particles.

Reliable Western blot protocol:

This protocol has been routinely used for western blot membranes measuring 10 cm x 10 cm (miniblot), 10 cm x 20 cm (midiblot) and 20 cm x 20 cm (maxiblot). 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. We recommend a total of 3 washes here.
   
   
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 a method of detection compatible with your choice of secondary antibody component. For peroxidise detection method of choice (enhanced chemiluminescence was used in experiments displayed on this site). Capture data using either X-ray film or a CCD camera.
   
   

* 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.