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Learn from the experts. We know that western blotting protocols vary depending on your particular laboratory set-up and reagents of choice. Here are a series of protocols, each explicit to a specific combination of reagents, detection modes, and laboratory equipment.
to the blot for 1–3 hr at room temperature depending on antibody quality and performance. 5. Rinse the blot 3–5 times for 5 min with TBST. 6. Incubate in the HRP-conjugated secondary antibody solution for 1 hr at room temperature. Note: The antibody can be diluted using 5% skim milk in TBST. 7. Rinse the blot 3–5 times for 5 min with TBST.
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- Western Blot Video: SDS-PAGE Separation of Proteins
This western blot protocol provides a general procedure for use with the majority of Bio-Rad reagents. In some cases specific recommendations are provided on product datasheets, and these methods should always be used in conjunction with product and batch specific information provided with each vial. Please note that a certain level of technical sk...
Appropriate controls should always be carried out. It may be useful to include a sample in which no primary antibody is used at all, in order to determine any nonspecific binding of the secondary reagent to the target tissue. Please contact Bio-Rad’s Technical Services Departmentto learn about recommended secondary reagents for specific application...
This video demonstrates SDS-PAGE separation of proteins using the Bio-Rad Comparative Proteomics Kit II: Western Blot Module. Assembly of the blotting sandwich and electroblotting are shown along with the steps for protein detection using a colorimetric assay. View Our Detection of Phosphorylated Proteins by Western Blotting Protocol
This protocol describes Bio-Rad’s Stain-Free Western Blotting Workflow, which takes only 5 hours to complete versus 16 hours for traditional western blotting. When following the lengthy and laborious traditional western blotting workflow, researchers assess the adequacy of their western blot data at the final step of the procedure.
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Learn more about western blotting techniques. Find step-by-step protocols and helpful tips on equipment, membranes, transfer conditions, and detection methods.
As highlighted by Figure 1 below, the Western blotting procedure relies upon three key elements to accomplish this task: the separation of protein mixtures by size using gel electrophoresis; the efficient transfer of separated proteins to a solid support; and the specific detection of a target protein by appropriately matched antibodies.
Bio-Rad has developed the V3 Western Workow utilizing stain-free technology to address the major concerns associated with the traditional western blotting procedure. This protocol describes the V3 Western Workow affording faster, more transparent, quantitative, and more reliable western blotting.
