Western blot | ECL development

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Lab. Bioclock & Aging/Dewan Md. Sumsuzzman

 ECL development

Protocol

  • Remove ECL Western Blotting Reagent Kit (Pierce® ECL plus western blotting substrate) from the refrigerator.
  • Prepare the substrate working solution by mixing Substrate A and Substrate B in a 40:1 ratio (example: 12 mL substrate A + 300 µL substrate B for 8 × 12 cm2 membrane). Use 0.125mL working solution per cm2 of membrane.

Note: The working solution is stable for up to 1 hour at RT.

  • Place the membrane on the other suitable clean surface, remove the water, add Pierce® ECL plus western blotting substrate (working solution) and spread evenly over the membrane surface.
  • Incubate blot with working solution for 5 minutes at RT.
  • Remove blot from working solution and place it in a plastic sheet protector or clear plastic wrap. Use an absorbent tissue to remove excess liquid and carefully press out any bubbles from between the blot and the membrane protector.
  • Place the protected membrane in a film cassette with the protein side facing up. Turn off all lights except those appropriate for X-ray film exposure (e.g., a red safelight).

Note: Film must remain dry during exposure. For optimal results, perform the following precautions:

  • • Make sure excess substrate is removed from the membrane and the membrane protector.
  • • Use gloves during the entire film-handling process.
  • • Never place a blot on developed film, as chemicals on the film may reduce signal.
  • Cut the X-ray film up to the membrane size (fold the top of the X-ray film and mark the direction).
  • Carefully place X-ray film on top of the membrane. Perform a first-time exposure of 60 seconds. Vary the exposure time to achieve optimal results.

Note: Light emission is most intense during the first 5-30 minutes after substrate incubation. Light emission continues for several hours but decreases with time. Longer exposure times may be necessary as time elapses.

Caution: Any movement between the film and membrane can cause artifacts on the film.

  • Develop film using appropriate developing solution and fixative. If the signal is too intense, reduce exposure time or strip and reprobe the blot with decreased antibody concentrations.
  • Rinse the X-ray film thoroughly with flowing water and air-dry. Be careful that the water does not stick together.

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