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Chemiluminescent Western Blot

Secrets to Chemiluminescent Western Blotting

What you need to know to optimize your chemiluminescent Western blots Chemiluminescence remains the most frequently used method to detect target proteins on Western blots. Many reagents are commercially available for chemiluminescent detection but all share basic characteristics. The secondary antibody is labeled with an enzyme, usually horseradish peroxidase (HRP). After incubation with the secondary […]

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effect of blocking agent

Getting Rid of the Noise: Western Blot Blocking

Why is the Western blot blocking step important? After a sample has been transferred from the gel to a blotting membrane, most Western blotting protocols include a blocking step before incubating the blot with the primary antibody. Understanding how blocking works is key to maximizing the quality of your Western blot, improving specificity, sensitivity, and […]

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Western Blot Normalization

Quantitative Westerns: What is the best way to normalize your Western blot?

Far from being an “is-it-there-or-not” technique, modern digital detection instruments can make Western blotting reproducible and quantitative. By working within the linear dynamic range of your detection method and normalizing the data to control for variations in protein load and membrane transfer, you can get truly quantitative results. But what is the best way to […]

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High background when fluorescent western blot stained with ponceau

Problems with Ponceau? Consider Alternative Total Protein Stains for Fluorescent Western Blots

Ponceau S is a fast, reversible protein stain often used to confirm that protein samples have successfully transferred from the gel to the membrane before a researcher moves ahead with the time-consuming process of immunoblotting. Staining the membrane after transfer makes it possible to quickly and easily identify any problems such as incomplete or uneven […]

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Western Blotting