Western blot immunodetection

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. 

Figure 44-6 Western blot analysis of endogenous proteolytic fragments of cTn antibodies. (Prom KatrukhaAG, Bereznikova AV, Ftitaov VL, Esakova TV, Kolosova OV, Pettersson K, et al. Degradation of cardiac troponin I implication for reliable immunodetection. Clin Chem 1998 44 2433-40.)...

Check the purity of the purified protein with native and SDS/ PAGE gel electrophoresis with silver staining detection (Fig. 2a), followed by Western-blot and immunodetection of the product (Fig. 2b). 

Fig. 2 (a) Silver stain of gel electrophoresis of pure recombinant rat PAP (size in kDa). Lane 1 native PAGE. Lane 2. SDS-PAGE. (b) Immunodetection (size in kDa) of purified recombinant rat PAP by Western-blotting. Lane / native PAGE. Lane 2. SDS-PAGE. Immunodetection with rabbit polyclonal antibody against an N-terminal peptide of rat PAP (in house antibody) [3]... 

Most MAbs or polyclonal antibodies used in immunodetection procedures are either mouse or rabbit antibodies. With the exception of mouse IgG subclass 1, all these antibodies can be coupled to protein A-sepharose, allowing maximal correct orientation of the antibody for immunoprecipitation. The use of the CTOSslinker dimethyl pimelimidate (15) also means that the antibody remains attached to the protein A beads during electrophoresis producing cleaner Western blots. 

Fig. 5. Immunodetection of cytokinin oxidase from Zea mays (lanes 1 and 2) and wheat seeds (lane 3) on a Western blot, using antibodies to Zea oxidase. Rabbit IgG was detected by gold-labelled goat anti-rabbit second antibody with silver enhancement...

That PrP and PrP are not totally identical has been proven by their different sensitivity to proteolysis upon mild proteinase K treatment, PrP is totally hydrolyzed in amino acids, whereas under the same conditions PrP is partially resistant to the protease. This finding is important for two reasons first, it proves that PrP and PrP have distinct properties that would be impossible to explain if they were totally identical and second, it allows differentiation between PrP and PrP with a simple assay based on a routine Western blot analysis of brain extracts that either are or are not incubated with proteinase K prior electrophoresis, transfer on nitrocellulose, and immunodetection with an anti-PrP antibody. The principle of this assay is described in Fig. 9.3. 

Alba FJ, Daban JR. Nonenzymatic chemiluminescent detection and quantitation of total protein on Western and slot blots allowing subsequent immunodetection and sequencing. Electrophoresis 1997 18 1960-6. 

FIGURE 9.3 Immunodetection of PrP and PrP in brain extracts. Western immunoblot of brain homogenates from iminfected and prion-infected animals (e.g., Syrian hamsters). Samples were either not digested (-) or digested (+) with proteinase K for 30 min at 37°C prior to polyacrylamide gel electrophoresis (PAGE). After electrotransfer, the blot is developed with a polyclonal rabbit anti-PrP antiserum that recognizes both PrP and PrP . PrP (blue band) is completely hydrolyzed under these conditions, whereas PrP (red band) is partially resistant to the protease treatment. In the third lane, the purple spot represents a mixture of PrP and PrP that are codetected in brain extracts from infected animals. 

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