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.
Here you can download a pdf version of the standard protocol for Western blot.
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
Protein samples (selected tissue lysates, cell lysates or over-expression lysates) are mixed with Laemmli buffer (to a final loading conc. of 2% SDS, 10% glycerol, 0.002% 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.
- 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.
- The proteins are transferred from the gels to PVDF membranes through semi-dry transfer using Trans-Blot® Turbo transfer system (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol.
All incubation and washing steps are performed at room temperature and with agitation.
- Dried membranes from 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.
- 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.
- To remove residual primary antibody, the membranes are washed 3 x 5 min in TBST (TBS with 0.1% (v/v) Tween20).
- 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.
- To remove residual secondary antibody, the membranes are washed 4 x 5 min in TBST.
- The membranes are incubated with detection reagent (Immobilon Western Chemiluminescent HRP Substrate, Millipore Corporation, Billerica, MA, USA) for 1 min.
- The image is captured using a CCD camera.