Practice question

Western blotting immunoblotting combines SDS-PAGE separation proteins molecular weight antibody detection achieving high specificity confirmatory test protein expression. Lysate preparation RIPA buffer detergents 0.1 percent SDS anionic 1 percent Triton X100 nonionic 0.5 percent deoxycholate salts 150 mM NaCl protease inhibitors PMSF aprotinin leupeptin preventing degradation sonication shearing DNA. Proteins denatured 95C 5 min Laemmli sample buffer SDS conferring uniform negative charge mass ratio 1.4 g/g beta-mercaptoethanol 2 percent reducing disulfides glycerol density bromophenol blue tracking. Loaded discontinuous polyacrylamide gradient 4-20 percent separating gel pH 8.8 stacking pH 6.8 electrophoresis 100V where proteins migrate inversely log MW separating 10-250 kDa. Proteins electrotransferred nitrocellulose 0.45 micron PVDF 0.2 micron hydrophobic activated methanol via semi-dry tank wet transfer electric field 100V 1h 4C preserving spatial pattern facilitating immunodetection. Membrane blocked 5 percent nonfat milk TBS Tween blocking nonspecific adsorption primary antibody rabbit mouse specific epitope p53 DO1 beta-actin AC15 HIV gp120 incubated overnight 1:1000 followed washes HRP-conjugated secondary goat anti-rabbit mouse recognizing Fc. ECL substrate luminol oxidized HRP H2O2 generating photons captured X-ray film CCD camera band intensity densitometry quantified antigen amount. Name western coined Burnette 1981 analogous Southern Edwin Southern DNA