Pierce™ ECL Western Blotting Substrate, 250 mL - FAQs

How can I increase the sensitivity of my Western blot?

Consider transferring to a different membrane or using a different detection method. We have observed increased sensitivity when using PVDF membranes in place of nitrocellulose. On PVDF membranes, using as little as 1 µg of total rat brain protein, PKC can be detected with alkaline phosphatase-mediated chromogenic detection in some cases using affinity-purified antibodies at a concentration of 0.5 µg/mL. Detection sensitivity can also be increased by using chemiluminescent detection, especially when using a SuperSignal West enhanced chemiluminescence subtrate (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/detect-proteins-western-blot/western-blot-detection-reagents/detection-technologies-western-blotting/chemiluminescent-western-blot-detection/supersignal-chemiluminescent-substrates.html) such as SuperSignal West Pico PLUS, SuperSignal West Dura, or SuperSignal West Femto. The secondary antibody should be used as recommend by the manufacturer and optimized as needed.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.