Proteases, if contaminating the experimental protein sample, may cause lysis or breakdown of the proteins (proteolysis). For example, if you are talking to a labmate during protein extraction and a speck of your saliva or air droplets, which contains proteases, might get into your sample and then degrade the polypeptides. Proteases might also get into your sample by accidental contamination. For example, your protein sample is from a living tissue and proteases are there in most cells. Proteases might be in your sample from the beginning. Proteases are enzymes that breakdown proteins. If the polypeptide chain is broken down or truncated, Western blot results will be erroneous.įor this, during protein extraction and isolation steps, great care needs to be taken that the polypeptides are not broken down by the action of proteases. However, it is critically important that the entire polypeptide chain for all proteins in all of the samples that will be Western blotted are preserved and protected from breaking down. Depiction of where protein samples my originate from for extraction and analysis.įor some insight into common approaches for protein extraction and isolation from the source its sample (cell line, tissue, microbial culture etc.) please refer to this article.įor Western blot analysis, proteins in the sample, in most experiments, are denatured to their primary structure by the action of heat, the detergent Sodium Dodecyl Sulfate (SDS), and reducing agents such as beta mercaptoethanol (BME) or dithiothreitol (DTT). For example, a clinical scientist might want to know whether a specific protein is present in the patient’s blood or in an excised tumor.įigure 1. 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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