Western blot results ncbi7/30/2023 ![]() Here, we describe a modification of the incubation method that overcomes this limitation without sacrificing sensitivity.ĭuring incubation of the membrane, antibodies float in solution while antigens are immobilized on the membrane. Especially, long incubations of antibody with membranes are required. Although western blot is performed routinely, it is a time-consuming and labor intensive method. Combined with digital image analysis, this method can reliably analyze the abundance of proteins and protein modifications 2. This technique is used to examine the presence, relative abundance, relative molecular mass, post-translational modification of proteins 1. Western blot (also known as Immunoblot) is a powerful and fundamental technique across a broad range of scientific and clinical disciplines. This simple protocol allows researchers to better explore the analysis of protein expression in many samples. This method can be applied to western blots using both chemiluminescent and fluorescent detection. The resulting ultra-high-speed western blot can be accomplished in 20 minutes without any loss in sensitivity. Combination of the CDR method with a commercial immunoreaction enhancing agent boosted the output signal and substantially reduced the antibody incubation time. We also utilized an immunoreaction enhancing technology to preserve the sensitivity of the assay. Here, we present an innovation that dramatically reduces these incubation intervals by improving antigen binding via cyclic draining and replenishing (CDR) of the antibody solution. These incubation steps are required due to slow diffusion of antibody from the bulk solution to immobilized antigen on the membrane: antibody concentration near the membrane is much lower than the bulk concentration. However, the western blotting procedure famously takes hours, even days, to complete, with a critical bottleneck being the long incubation times that limit its throughput. This technique has a rich history and remains in widespread use due to its simplicity. It is commonly used to determine the relative size and abundance of specific proteins as well as post-translational protein modifications. Western blot (also known as an immunoblot) is a canonical method for biomedical research.
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