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.
![Protein-labeling_1600x1400_v1 - VectorLabs - VectorLabs Protein Labeling X V Protein Labeling X V Protein labeling x v](https://vectorlabs.com/wp-content/uploads/2022/11/Protein-labeling_1600x1400_v1.webp)
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).
![Stylized-graph_1600x1400_1 - VectorLabs - VectorLabs Stylized Graph X Stylized Graph X Stylized graph x](https://vectorlabs.com/wp-content/uploads/2022/11/Stylized-graph_1600x1400_1.webp)
Characterization
Purify labeled antibodies and proteins using affinity chromatography with agarose-immobilized, Ig- or biotin-specific reagents.
![western-blotting_1600x1400_1 - VectorLabs - VectorLabs Western Blotting X Western Blotting X Western blotting x](https://vectorlabs.com/wp-content/uploads/2022/11/western-blotting_1600x1400_1.webp)
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.
![Fl-antigen_1600px-1600x1400_2 - VectorLabs - VectorLabs Fl Antigen Px X Fl Antigen Px X Fl antigen px x](https://vectorlabs.com/wp-content/uploads/2022/11/Fl-antigen_1600px-1600x1400_2.webp)
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.
![Western-blot-fl1600x1400_1 - VectorLabs - VectorLabs Western Blot Flx Western Blot Flx Western blot flx](https://vectorlabs.com/wp-content/uploads/2022/11/Western-blot-fl1600x1400_1.webp)
Visualization
If using an enzymatic detection method, visualize the labeled antibody with an appropriate substrate for chromogenic or chemiluminescent/ chemifluorescent detection.