Western blotting is a very reliable method to measure protein quantity and provide protein identification using antibodies and molecular weight (MW). Other methods for protein quantification, such as Bradford assays and UV-Vis spectroscopy, are not as specific or sensitive as Western blotting. They can also be affected by the presence of other substances in the sample, such as detergents and salts.
Western Blotting combines the following key features:
Specificity: Western blotting uses antibodies to specifically bind to the target protein, even in complex mixtures of other proteins. This is essential for accurate protein identification and quantification. The selection of the correct primary antibody is critical to obtaining accurate experimental results.
Sensitivity: Western blotting can detect very small amounts of protein, down to pico-gram levels. This is important for studying proteins that are expressed at low levels.
Versatility: Western blotting can be used to detect a wide range of proteins, including modified proteins, phosphorylated proteins, and protein complexes.
How the BlotCycler helps
The BlotCycler is a semi-automated Western blotting system that can improve the reliability and reproducibility of Western blot results. It does this by precisely controlling the timing, and mixing of all the different steps during Western blot processing protocol that include: Blocking, Primary & Secondary Incubation, washing and recycling of the primary antibody.
Once the BlotCycler protocol is finished standard Chemiluminescene can be used to perform quantitative analysis to measure the intensity of the protein bands on the blot. This allows researchers to compare the expression levels of the target protein in different samples.
Overall, Western blotting is the most reliable method for measuring protein quantity and providing protein identification because it is specific, sensitive, and versatile. The BlotCycler can further improve the reliability and reproducibility of Western blot results by precisely controlling the different steps in the protocol.