WesternSure® PREMIUM Chemiluminescent Substrate

Components (926-95000)

WesternSure PREMIUM Luminol Enhancer Solution: 50 mL

WesternSure PREMIUM Stable Peroxide Solution: 50 mL

Applications

WesternSure PREMIUM Chemiluminescent Substrate is a highly sensitive, enhanced substrate for detecting horseradish peroxidase (HRP) on immunoblots. The Substrate is optimized for use with the C‑DiGit Blot Scanner, Odyssey XF Imager, Odyssey XF Imager, and Odyssey M Imager (Model 3350).

WesternSure PREMIUM Chemiluminescent Substrate is designed for high sensitivity and long signal duration, making it ideal for use with imaging systems and film.

Specifications

  • Storage: Room temperature

Protocol

Optimize the antigen and antibody concentrations as well as blocking conditions for each experimental system. WesternSure HRP-conjugated secondary antibodies (LI-COR P/N 926-80010 or 926-80011) are designed for use with this substrate. HRP-conjugated secondary antibodies from other vendors may be suitable. Optimization may be required.

Avoid buffers containing sodium azide, as it will inhibit HRP activity.

  1. Dilute the primary antibody per vendor recommendations.

  2. Dilute WesternSure HRP Secondary Antibody (1:5000-1:100,000).

  3. Prepare the Working Solution. Mix equal parts of Stable Peroxide Solution and Luminol Enhancer Solution. Use 0.1 mL Working Solution per cm2 of membrane.

    Working Solution is stable for 8 hours at room temperature.

  4. Incubate the blot for 5 minutes in the Working Solution.

  5. Drain excess reagent.

  6. Capture the digital image on the C‑DiGit Blot Scanner, Odyssey XF Imager, Odyssey XF Imager, or Odyssey M Imager or expose the blot to film.

Protocol for Digital Imaging

  • C‑DiGit Blot Scanner: Place the wet blot directly on the scanning surface with the protein side face down. Place a plastic sheet over the membrane to prevent it from drying. Select ‘Standard’ or ‘High’ sensitivity setting in Image Studio™ Software, then start the scan.
  • Odyssey XF Imager or Odyssey XF Imager: Place the wet blot in a clear plastic sleeve to prevent it from drying. Place the blot in the imaging tray with the protein side face up then return the tray to the imaging drawer. Select and follow the Membrane workflow in LI‑COR® Acquisition Software, then select the acquisition time and start the scan.
  • Odyssey M Imager: Prepare the blot and add substrate away from the imager's Scan Surface. Follow instructions in the Odyssey M manual licorbio.com/m-manual. Select the Membrane workflow in LI‑COR® Acquisition Software, then follow the steps in the workflow.

Protocol for Film

  • Cover the blot with clear plastic wrap or a clear plastic sleeve. Place the blot in a film cassette with the protein side face up. Place film on top of the blot and expose it for the desired length of time.