Western blot blotting protocol

Two principal methods are available (1) western blotting using phos-phospecific antisera and (2) isoelectric focusing followed by western blotting. Protocols based on mass spectroscopic methods may also be valuable, especially where either the phosphorylation site has not been identified or the protein contains multiple sites of phosphorylation. 

ECL western blotting protocols Amersham International pic. Bucks, UK. 

ECL Western Blotting Protocols (1991) Amersham International pic, Amersham, UK. 

A modified Western blotting protocol has been developed that increases the binding specificity of antigens and antibodies without... 

Li, W., Murai, Y., Okada, E., Matsui, K., Hayashi S., Horie, M., and Takano, Y. (2002) Modified and simplified western blotting protocol use of intermittent microwave irradiation (IMWI) and 5% skim milk to improve binding specificity. Pathol. Int. 52, 234-238. 

Holmes JL, Pollenz RS. 1997. Determination of aryl hydrocarbon receptor nuclear translocator protein concentration and subcellular localization in hepatic and nonhepatic cell culture lines development of quantitative Western blotting protocols for calculation of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein in total cell lysates. Molec. Pharmacol. 52 202-11... 

Both methods have been validated according to the Japanese validation protocol (Sakai et al., 2008), and both primers are commercially available. All the Western blotting and PCR kits are shown in Table 4.11. 

After the run common fixation and staining protocols are used. For Western blotting according to the Kyhse-Anderson semidry protocol (Protocol 2.4.3), the following buffers are recommended by ScHAGGER and v. Jagow ... 

Identify the ligate by specific antibodies as described in Protocol 2.5.4 (Western blot). 

Identify the protein spots as usual, i.e., by staining, autoradiography, gel overlay, or Western blot. In the latter case the gel must be separated from the GelBond foil prior to electrotransfer. For this purpose a film remover (Gorg 2003, Fig. 19) is used The gel is placed on the cylindrical remover with foil down, clamped on an edge, and a thin stainless steel or nylon wire is pulled between foil and gel towards to your body. Cover the gel with the wetted blotting membrane (cf Protocol 2.4.3) and transfer membrane as well as gel to the blotting apparatus. 

Protein bands were detected with the ECL plus Western Blotting Detection system according the manufacture s protocols. 

Polyclonal or monoclonal phage antibodies can be used as reagents to detect antigens on Western blots (see Chapter 20). Here we describe the detection of purified antigen, but the same protocol can be used for antigen present in complex protein mixtures. 

The protocol involves a classical SDS-PAGE (10% polyacrylamide) run, followed by transfer onto a Western blot membrane and immunodetection with an anti-pIII antibody. Nevertheless, special care must be taken during sample preparation, because phages are very stable and difficult to denature. The protocol is similar to typical SDS-PAGE sample preparation, except that / -mer cap toe thanol should be replaced by fresh dithiothreitol (DTT, 5 mM final concentration), and the samples should be boiled in a water bath for at least 15 min. Moreover, because the pIII-fusion protein is a minor component of the virion, a large amount of phages should be loaded onto the gel, typically around 1012 phages per lane. 

A standard double-sandwich enzyme-linked immunosorbent assay (ELISA) or Western blot analysis is used. As the concentration of factor is very low in normal plasma (approximately 0.1 mg/1 for factor VIII), it is necessary to subject plasma samples to cryoprecipitation in order to concentrate the sample, prior to Western blot analysis. The cryoprecipitation protocol described by Bi et al. is as follows (Bi et al., 1996 Sarkar et al., 2000 Mah et al., 2003). Plasma samples are collected as described above for the Coatest assay. Plasma is then frozen at -80 °C overnight. Frozen samples are then subject to centrifugation at 7000 x g for 20 min at 4°C. The precipitate is washed with... 

Alterations of the mitochondrial membrane may precede those that occur in the nucleus. These changes can involve both the inner and the outer membrane, leading to a dissipation of the transmembrane potential and/or to the release of intermembrane proteins through the outer membrane. The main group of proteins responsible for mitochondrial alterations consist of the proteins known as Bax, which form pores in the outer membrane, causing the release of cytochrome c to the cytoplasm (Loeffler and Kroemer, 2000 Arden and Betenbaugh, 2004). Western blot techniques can be used to specifically detect the presence of cytochrome c in the cytoplasm of apoptotic cells. However, complex purification protocols are required, and there is the possibility of incomplete separation of mitochondria from the cytoplasm therefore, this technique is not very popular. 

Unlike ELISA, which can detect and quantitate host-cell proteins as a group, Western blots detect single protein impurities. Western blot analysis starts with an SDS-PAGE protocol but does not include a final colorimetric staining step. Instead after electrophoresis the proteins are electrotransferred (blotted) from the gel onto a thin membrane. Membranes made of nitrocellulose or PVDF are most often used. Once the transfer is complete, the membrane is incubated with a nonspecific protein solution to saturate and to... 

Protocol 12 Western Blot Analysis after Derivatization with Sanger s Reagent... 

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