

What is Western Blotting?
Your common Western blotting questions, answered. Western blotting is a widely used analytical technique that can identify one or more specific proteins in a complex
Chemiluminescence is a popular indirect detection method for Western blotting. This technique is very good at answering the question, “Is my protein there or not?” and can provide a qualitative answer to the question “Is the amount of my protein different between these two samples?”
In chemiluminescent detection, the primary antibody binds to the target protein on the membrane, and the location of the primary antibody is detected using a secondary antibody conjugated to an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP).
A substrate for the enzyme is added and when the enzyme acts on the substrate, light is emitted (Figure 1.6). The light can be detected using a CCD camera or x-ray film (Figure 1.7). The sensitivity of detection depends on the choice of substrate—commercially available substrates for HRP can detect proteins in the femtogram range.
Chemiluminescent detection is often used because it is specific, easy to perform, and highly sensitive. The labeled secondary antibody can be used across multiple experiments to detect any primary antibody of the correct species, making the approach cost-effective.
Flexible gel doc imaging system for Western blotting and DNA gels
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Your common Western blotting questions, answered. Western blotting is a widely used analytical technique that can identify one or more specific proteins in a complex
One of the most common questions when troubleshooting problematic Western blots is, “Why is the background so high?” High or uneven background doesn’t just look bad-
Validating your antibodies, i.e. confirming that an antibody recognizes your protein of interest with low cross-reactivity to other targets, is critical for ensuring consistent, reproducible
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