However, the number of antibodies available for Western blotting is expanding at a rapid pace as the production costs have decreased. The main disadvantage of Western blotting is that this technique requires a specific antibody to a target protein thus many protein targets cannot be investigated because of the lack of specific antibodies. This method offers many advantages over other techniques for detecting and semi-quantifying target proteins, allowing the detection of a single target out of thousands of proteins as well as obtaining molecular weight information about the target protein in the same experiment. Western blotting is a technique that was developed in 1979 and is now a commonly used technique in biomedical research. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Learn more about how our radiation safety products work.Western blotting is a commonly used technique in biological research.UVITEC Chemiluminescence Documentation Systems.Blue Light Transilluminators BEST SELLER.Gel Documentation Systems Package Deals SAVINGS.Including details of all the relevant equipment and consumables that we offer. You can learn more about how to perform western blots in our handy application note. Learn more about Western Blotting in our Application Note.Western Blot Imagers Package Deals SAVINGS.powerPRO 5 Outlet Power Supplies Best Seller.Clinical and Pharmaceutical Electrophoresis. Denaturing Gradient Gel Electrophoresis Equipment.Vertical Electrophoresis Systems (PAGE).Horizontal Gel System Package Deals SAVINGS.multiSUB ClearSight Condensation Free Systems.multiSUB Horizontal Gel Systems Best Seller.Horizontal Electrophoresis Systems (Agarose).
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