Application Overview

Chemiluminescent Western Blotting

Western blotting detection with various chemiluminescent substrates can also be done using the Odyssey^® Fc.

LI-COR Biosciences provides protocols, application notes, helpful tips and references to help you produce high-quality Western blotting results.

Chemiluminescent Western FAQs

Here are some frequently asked questions about chemiluminescent Western blots and the Odyssey Fc Imager. For more FAQs on chemiluminescent Western blotting and other information and protocols, visit biosupport.licor.com.

• Can I dilute the HRP-conjugated secondary antibodies in the Odyssey^® blocking buffer?

  • No. Odyssey blocking buffer contains sodium azide as a preservative. Sodium azide binds irreversibly to the HRP enzyme, inhibiting the binding of the substrate and slowing the chemiluminescent reaction. This results in less light production that may affect the appearance of less intense bands or even the entire blot. For optimal results do not use any solutions containing sodium azide for chemiluminescent Western blotting.



• Can I use milk-based blockers?

  • Yes. Milk-based blockers can be used for chemiluminescent detection but should be avoided when detecting phosphoproteins or glycoproteins. Milk-based blockers may contain endogenous biotin and glycoproteins, resulting in higher background on the membrane.



• What is the best blocker for chemiluminescent Western blots?

  • It is best to try several blockers to find the one that gives the most satisfying data for each antigen and antibody pair. There is not a best blocker for all conditions.



• Why is the signal missing in the middle of the bands?

  • Too much secondary antibody on the membrane results in consumption of all the substrate in that area. Without substrate, there is no chemiluminescent signal and a white spot appears in the center of the band. Try different dilutions of the primary and secondary antibodies to find what gives the best results, or try changing the substrate.



• Does it matter how I wash the membranes after antibody incubation?

  • Yes. Adequately washing the membranes will greatly improve the appearance of the chemiluminescent Western blot. Wash the membranes with a saline-buffered solution containing 0.05 to 0.1% of a non-ionic detergent such as Tween^® 20. Wash four times for five minutes each time with ample wash solution on a shaker or rotator.



• How do I apply the substrate?

  • Make sure the substrate is at room temperature before use. Apply carefully and avoid pooling to prevent splotches and areas of high background. Carefully wick off any pools of substrate before imaging.



• Can I wrap the blot in plastic wrap before imaging?

  • Wrapping the blot in plastic wrap may cause unwanted background, especially if it is folded or handled roughly. If using plastic wrap it is important to avoid wrinkles as they scatter light, resulting in high background. You can also image the plastic wrap alone first to determine if the plastic itself scatters light. If it does, try different brands of plastic wrap to find the best one.



• The membrane dried during imaging. Can I apply more substrate and image again?

  • No. Applying more substrate to a dried blot will likely result in high background.

Support Materials

• Optimizing Chemiluminescent Western Blots

• Chemiluminescent Western Blots - Frequently Asked Questions