Or, the samples might also come from a cell or microbial culture. Samples for Western blot analysis may come from plant or animal tissue, for example, to study whether a specific protein is being expressed in a transgenic spinach leaf or in the mouse ear. Since then, other developed blotting techniques have been named for the cardinal directions, just for keeping synchrony in the nomenclature of blotting techniques: Northern for RNA and Western blotting for protein analysis respectively. Western blot got its name from another, earlier discovered blotting technique called Southern blot (for DNA analysis) named for its discoverer Edwin Southern. Probing the membrane with primary and secondary antibodiesįinal detection assay: developing the immunoblot for visual analysis The actual detection process in a Western blot Some variations in Western blot will also be discussed.Ī note about immunoblot (Western blot) membranes In this article, we will look at the theoretical concepts underlying each step in a Western blot. Western blot plays a big role in protein biochemistry research. Indeed, protein analysis is a cornerstone in modern bioscience. Developing the blot formed on the membrane in order to visually analyze the results.Detecting the target protein of interest on the membrane using one or more antibodies.Transferring the proteins from the gel to a membrane.Extracting proteins from the experimental samples – plant/ animal tissue or cell line or microbial culture.The transfer of the proteins from the gel to the membrane gives the name “blot,” just as spilled juice is blotted off from your dining table using a paper towel.īasic steps of a Western blot experiment are: Finally, the membrane is developed using detection reagents for visual comprehension of the results. Western blot involves resolving out the proteins in the experimental sample by gel electrophoresis, transferring the electrophoresed proteins from the gel onto a membrane, and then detecting the target protein of interest immobilized on the membrane using antibodies that act as molecular probes. Western blot, also called immunoblot, is a commonly used technique for qualitatively and quantitatively analyzing proteins in an experimental sample.īasically, Western blot tells you whether your protein of interest is present in the sample and by how much in relative abundance.
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