![]() When lysing tissues or cells, you should prepare samples containing similar total protein concentrations as determined by a protein assay kit. This will allow for appropriate comparison of the expression of proteins involved in apoptotic signaling. In the case of diseased or cancerous tissue, a healthy or benign sample should be used for comparison. When working with a cell line, any treatment that has been applied must be compared to an appropriate untreated set of cells. This validation may take several forms including verification with a second antibody, target protein knock out/knock down studies or demonstrating a well-known change in expression following a particular experimental treatment.Īs in any apoptosis assay it is important to ensure appropriate controls are included. However the antibody will need to be validated in your particular experimental conditions. Detecting a band of an appropriate molecular weight is the first step in a successful and verified western blot.Įxperimental controls and sample preparationīio-Rad provides highly specific validated antibodies. This information will help when interpreting the results. When preparing for western blot, there are several factors about your target protein to be aware of, including the localization of your protein, the molecular weight and its level of expression. The choice of target proteins will depend on the particular area of apoptosis you wish to study. For IP specific products, protocols, tips and tricks, refer to our immunoprecipitation application page. Therefore in addition to basic western blotting, immunoprecipitation (IP) is also a highly useful technique for detecting proteins contained within a complex. The main markers of apoptosis detectible by western blot include phosphorylated B-cell lymphoma 2 (Bcl-2) family members, activated fragments of caspases and cleaved poly (ADP-ribose) polymerase-1 (PARP-1).Īpoptosis relies on the formation of a number of protein complexes (such as the apoptosome and the death induced signaling complex (DISC) amongst others). We have included some general considerations on western blotting and detailed information on how western blot can be used to analyze the various stages of apoptosis. Here we offer advice and information on analyzing apoptosis by western blotting. Western blotting allows for the detection of proteins (and their PTMs) with far greater versatility and specificity than other antibody based techniques. Post translational modifications (PTMs) such as phosphorylation are also common events within apoptotic signaling. There are many proteins whose change in expression or localization is indicative of a particular point in the apoptotic process. Western blotting analysis of apoptosis allows for the detection, localization and quantification of proteins involved in apoptotic signaling. ![]()
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