Western Blot Standard Protocol

The protocol described below is the Atlas Antibodies standard protocol for Western blot, optimized for Triple A Polyclonals and PrecisA Monoclonals.

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Alternative BSA blocking protocol

The standard Western blot protocol described on this page uses a milk-based blocking buffer. If your antibody requires BSA blocking, please use this alternative Western blot protocol using BSA blocking instead.

Note: The BSA blocking protocol is the recommended protocol for the Anti-FOS antibody (AMAb91417) as well as the Anti-KLF4 antibodies (AMAb91388 and AMAb91389) for Western blot.

Electrophoresis and blotting

Sample preparation

Protein samples (selected tissue lysates, cell lysates, or over-expression lysates) are mixed with Laemmli buffer (to a final loading concentration of 2% SDS, 10% glycerol, 0.005% bromophenol blue, 0.0625 M Tris-HCl), supplemented with DTT to a final concentration of 50 mM, and incubated in 95°C for 5 min.

Procedure

1. Protein samples are loaded onto Criterion TGX Precast Gels, 4–20% polyacrylamide (Bio-Rad, Hercules, CA, USA). The electrophoresis is run according to the manufacturer’s protocols.
2. The proteins are transferred from the gels to PVDF membranes through semi-dry transfer using the Trans-Blot® Turbo transfer system (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol.

Immunodetection

All incubation and washing steps are performed at room temperature and with agitation.

Procedure

1. Dried membranes from the previous steps are activated in methanol for 20 seconds. To prevent non-specific background binding of the primary and/or secondary antibodies to the membrane, membranes are blocked in milk-based blocking buffer (5% (w/v) non-fat dried milk in TBS with 0.1% (v/v) Tween20) for 45 min.
2. The primary antibody is diluted in blocking buffer and incubated with the blocked membranes for 1 hour. 
   NOTE: The recommended working dilution of the primary antibody is to be considered as a guideline only. Optimal dilution must be determined by the user.
3. To remove residual primary antibody, the membranes are washed 3 x 5 min in TBST (TBS with 0.1% (v/v) Tween20).
4. The secondary antibody (for monoclonal antibodies: HRP-conjugated Goat Anti-Mouse Immunoglobulin; for polyclonal antibodies: HRP-conjugated Swine Anti-Rabbit Immunoglobulin, Dako, Glostrup, Denmark) is diluted 1:3000 in blocking buffer and incubated with the membranes for 30 min.
5. To remove residual secondary antibodies, the membranes are washed 4 x 5 min in TBST.
6. The membranes are incubated with a detection reagent (Immobilon Western Chemiluminescent HRP Substrate, Millipore Corporation, Billerica, MA, USA) for 1 min.
7. The image is captured using a CCD camera.