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PrEST Control Antigens
Primary Antibodies

 

Western Blot Protocol - BSA Blocking

The recommended Western blot protocol optimized for antibodies requiring bovine serum albumin (BSA) blocking is described below. 

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

 

Download Protocol in PDF

 

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, 0.002% 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.

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 wash 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 a bovine serum albumin-based blocking buffer (2% (w/v) in TBS with 0.1% (v/v) Tween20) for 45 min.
  2. The primary antibody is diluted in a blocking buffer and incubated with the blocked membranes for 1 h. 
    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.