![]() If your target protein is expressed at low levels, read our guide for detecting low abundant proteins which provides useful tip and tricks to boost target protein detection However polyclonal antibodies tend to produce a greater degree of non-specific binding than monoclonal antibodies. A polyclonal antibody may provide a greater signal than a monoclonal as a result of binding to multiple epitopes. For more information on sample preparation view the western blot sample preparation page.Ĭhoosing between a monoclonal or polyclonal antibody is an important step in antibody selection. When detecting the phosphorylation of a target protein by western blot it is crucial to prepare your samples on ice with the appropriate lysis buffer containing fresh phosphatase inhibitors. Phosphorylation is crucial to almost all cell signaling pathways including apoptotic signaling. For example detecting nuclear proteins such as transcription factors can be significantly enhanced by using a specialized ReadyPrep™ Protein Extraction Kit. Knowing the subcellular localization of your target protein is crucial as it may influence sample preparation. Bio-Rad’s Stain-Free Technology offers a total protein normalization that functions over a wider range of detection and is more sensitive and accurate than traditional house-keeping protein methods. However this process can often be over a narrow linear range. Traditionally this relative quantitation between two samples has relied on comparing the expression of a house-keeping protein to your protein of interest. This will make western blot relative quantitation easier and give a more reliable indication of the change in the target proteins expression during apoptosis. 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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