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Western Blot (WB) - Protocol

Western blot is a commonly used molecular biology technique that analyzes proteins of interest in samples. Cell lysates are generated from cell culture lysis and separated by weight in gel electrophoresis (SDS-PAGE). These proteins are then transferred from gel to membranes where they are blocked and probed with specific antibodies for detection of proteins of interest. With sensitive and specific antibodies, modified proteins or unmodified proteins are easily detected in small samples.

Protocol:

  • 1. Block membrane by incubating 1 hour at room temperature with shaking in Blocking Solution (5% nonfat milk in TBST (50mM Tris, 100mM NaCl, 0.05% Tween-20, pH 7.6).
    Note: Use 5% BSA in Blocking Solution for phospho specific antibodies.
  • 2. Dilute primary antibody at the appropriate dilution in Blocking Solution.
  • 3. Incubate the membrane with diluted primary antibody overnight at 40C with agitation.
  • 4. Remove antibody solution. Wash the membrane 3 times for 5-10 minutes each time at room temperature in TBST (50mM Tris, 100mM NaCl, 0.05% Tween-20, pH 7.6) with shaking.
    Note: Increasing the concentration of Tween-20 to 0.1% reduces the background and increases the specificity, but it will reduce the sensitivity.
  • 5. Incubate membrane with secondary AP or HRP conjugate diluted (according to manufacturer’s instructions) in Blocking Solution for 1 hour at room temperature with shaking.
  • 6. Wash the membrane as in Step 4.
  • 7. Wash membrane with TBS for 2-5 minutes before proceeding Chemiluminescent Reaction.
  • 8. Prepare and use the Chemiluminescent substrate (for AP or HRP) according to the manufacturer’s instructions.
  • 9. Immediately wrap the membrane and expose to X-ray films for 10 second to 1 hour period. The exposure time may vary according to the mount of antibody and antigen. Or place into imager and adjust accordingly.