Prestige Antibody® Western Blot Procedure

WB Protocol Revision B, currently used in our P&QC procedures, is an optimized version of the Human Protein Atlas (HPA) original protocol. The HPA original protocol can be found on our website in the Archive folder.

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 conc. of 2% SDS, 10% glycerol, 0.002% bromophenol blue, 0.0625 M TrisHCl), supplemented with DTT to a final concentration of 50 mM, and incubated in 95°C for 5 min.


  1. Protein samples are loaded onto Criterion TGX Precast Gels, 4–20% polyacrylamide (Bio-Rad, Hercules, CA, USA). The electrophoresis is run according to manufacturer’s protocols.
  2. 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 manufacturer’s protocol.


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


  1. 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 minutes.
  2. The primary antibody is diluted in blocking buffer and incubated with the blocked membranes for 1 hour.
    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 minutes 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 minutes.
  5. To remove residual secondary antibody, the membranes are washed 4 x 5 minutes in TBST.
  6. The membranes are incubated with detection reagent (Immobilon Western Chemiluminescent HRP Substrate, Millipore Corporation, Billerica, MA, USA) for 1 minute.
  7. The image is captured using a CCD camera.  

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