Western Blot

What is western blotting?

Western blotting is a popular technique used for protein detection and quantitation. It enables the separation and identification of a specific protein of interest from a complex mixture of proteins, for example a cell lysate. With applications in diagnostics, biotechnology, molecular biology, proteomics, and much more, western blots are widely relied upon to evaluate levels of protein expression in cells, as well changes in size and other properties.

Western blot methods

There are several western blot methods to consider depending upon the secondary antibody used, detection of the target protein may be colorimetric, chemiluminescent, or fluorescent. These methods are described below and require different equipment for detection. For example chemiluminescence can be detected using x-ray film or digital imaging equipment while a fluorescent secondary antibody requires a fluorescence imager. Each type of detection has advantages and disadvantages that need to be considered when selecting a method.

Another method that we have introduced the ScanLater™ Western Blot Detection System, enables first-of-its-kind western blot detection in a multi-mode microplate reader platform. Utilizing this substrate-free assay, researchers can achieve equivalent sensitivity to traditional chemiluminescence assays while consolidating western blot, ELISA, and other applications onto a single reader. Our featured application notes below provide further details.

ScanLater™ Western Blot Detection System enables western blot detection on microplate reader

Western blotting assay workflow

Here are the steps used for a typical western blot assay:

Western blot assay workflow

1. Perform electrophoresis – Proteins are first separated by size using gel electrophoresis.

2. Transfer – They are then transferred to a blotting membrane, usually made of nitrocellulose or PVDF, which is probed with a primary antibody specific to the protein of interest.

3. Block with blocking buffer – This prevents binding of the primary antibody (next step) to the membrane itself.

4. Incubate with primary antibody – Primary antibody binds to the target protein.

5. Wash with wash buffer – Unbound primary antibody is washed away.

6. Incubate with secondary antibody – A secondary antibody that recognizes and binds the primary antibody is added. This secondary antibody is conjugated to an enzyme or other material that allows detection of the protein of interest, which appears as a band on the blot.

7. Wash with wash buffer – Unbound secondary antibody is washed away.

8. Detect using method/chemistry of choice – Target protein can be detected by colorimetric, fluorescent, or luminescent methods, depending upon the secondary antibody used.

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