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.

Step 1

Labeling

Labeling

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).

Biotin and Digoxigenin Labeling Reagents
Step 2

Characterization

Characterization

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

Biotinylated Reagents Magnetic Beads and Agarose
Step 3

Blocking

Blocking

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.

Endogenous Biotin Blocking Kits Autofluorescence Quencher Western Blot Blocking Solutions
Step 4

Detection

Detection

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.

Fluorophore Conjugated Avidin/Streptavidin Enzyme Conjugated Avidin/Streptavidin ABC Kits for Western Blot Detection Enzyme Polymers for Western Blot Detection
Step 5

Visualization

Visualization

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

Enzyme Substrates