ABSTRACT
Western blot analysis is based on a protein-protein hybridization technique that is used for immune detection of speci c antigen(s) of interest in a complex mixture of proteins. This is a simple, sensitive, and effective technology that has been used in immunology, molecular and cellular biology, and protein chemistry. The principle of western blotting is as follows: (1) A protein mixture is rst separated according to molecular size using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). (2) The separated protein molecules are then immobilized onto a nitrocellulose or polyvinylidene di§uoride (PVDF) membrane. (3) The speci c protein band of interest is identi ed by use of a speci c antibody raised against a speci c antigen (protein), which can speci cally bind to the antigen (protein) of interest in the protein mixture that is immobilized on the membrane. (4) The antibody-antigen complex is then detected by an enzyme linked to a second antibody and substrate, or by the use of 125I-labeled protein A or its equivalent.1-4 The antigen can also be directly detected with a §uorescence-labeled antibody, which directly binds to the antigen
16.1 Separation of Proteins by SDS-PAGE and the Use of 2D Gels ........................................... 314 16.1.1 SDS-PAGE ............................................................................................................... 314
16.1.1.1 Preparation of the Separating Gel .............................................................. 314 16.1.1.2 Preparation of the Stacking Gel ................................................................. 315 16.1.1.3 Loading the Samples and Protein Standard Markers onto the Gel ........... 315 16.1.1.4 Electrophoresis ........................................................................................... 316
16.1.2 Two-Dimensional Gel Electrophoresis ..................................................................... 317 16.1.2.1 Preparation of the Focusing Gel (Range of pH: 4-6) ................................ 317 16.1.2.2 Loading the Samples onto the Gel ............................................................. 318 16.1.2.3 Electrophoresis ........................................................................................... 318 16.1.2.4 Post-Focusing Procedures .......................................................................... 319
16.2 Staining and Destaining of the Gel ...................................................................................... 319 16.2.1 Coomassie Blue Staining and Destaining Method ................................................... 319 16.2.2 Silver Staining Method ............................................................................................. 321
16.3 Transfer of Proteins from the Gel to Membranes ................................................................. 321 16.3.1 Wet Blotting .............................................................................................................. 321 16.3.2 Semidry Blotting ...................................................................................................... 322
16.4 Immunodetection of Speci c Protein(s) ............................................................................... 323 16.4.1 Alkaline Phosphatase ............................................................................................... 323 16.4.2 Chemiluminescence Detection ................................................................................. 325
16.5 Quantitative Analysis of Proteins after Western Blot Hybridization ................................... 325 16.6 Troubleshooting Guide ......................................................................................................... 326 16.7 Reagents Needed .................................................................................................................. 326 References ...................................................................................................................................... 330
in a protein mixture. After washing away the non-bound antibody, the antigen-labeled antibody can then be visualized under a microscope tted with a UV lamp that can excite the §uorescent tag using a speci c wavelength of light. However, the direct method needs a relatively large amount of the labeled antibody to obtain good detection, and the labeling of an antibody of particular interest is expensive. Therefore, the indirect immunodetection method is widely used today. The following detailed protocols are based on modi cations in the method of Towbin et al.2 who rst developed the technique.
