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Western Blotting Workflow

Western blotting is a technique that is often used in the purification, characterization, and identification of proteins. Performing a western blot involves separating proteins by gel electrophoresis and transferring the separated proteins to a blotting membrane. Once blotted, protein can then be detected either directly (using a labeled primary antibody) or indirectly (using a primary antibody and labeled secondary antibody).

Vector Laboratories supports your protein research by offering a wide range of reagents and kits for protein detection and analysis on western blots.

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Label antibodies with biotin (for avidin/streptavidin-based detection), peroxidase (HRP; for enzymatic, chromogenic detection), or fluorescent compounds (for fluorescence detection). The choice of label depends on many factors, including the sensitivity desired and instrumentation available for detection. Vector offers a variety of reagents and kits for protein labeling, as well as a wide range of prelabeled secondary antibodies (see Detection).

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Purify labeled antibodies and proteins using affinity chromatography with agarose-immobilized, Ig- or biotin-specific reagents.

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Prior to immunological detection, block unoccupied binding sites on the blot to prevent nonspecific binding of the detection antibodies. Failure to do so can lead to high background.

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Apply the labeled primary and/or secondary antibodies to the blot to detect the target protein. Choose from avidin/streptavidin-, enzyme-, or fluorescence-based detection methods and kits.

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If using an enzymatic detection method, visualize the labeled antibody with an appropriate substrate for chromogenic or chemiluminescent/ chemifluorescent detection.