Direct Immunodetection of Antigens Within the Precast Polyacrylamide Gel.

Western blotting is one of the few basic techniques widely used in the study of proteins in life science research. Despite its prevalence, the procedure has remained practically unchanged for more than 20 years. Although the method is viewed as being error-prone and as requiring excessive hands-on time, it is still widely accepted because it provides sensitive and direct information about the protein characteristics. The process is attractive to researchers because it reduces the investment in instrumentation and set up. Here we describe a procedure that eliminates the transfer step of western blotting and allows for antigen detection directly within the polyacrylamide gel, thus minimizing the investment necessary for setting up western blotting.

Direct Immunodetection of Antigens Within the Precast Polyacrylamide Gel.

Western blotting is one of the few basic techniques widely used in the study of proteins in life science research. Despite its prevalence, the procedure has remained practically unchanged for more than 20 years. Although the method is viewed as being error-prone and as requiring excessive hands-on time, it is still widely accepted because it provides sensitive and direct information about the protein characteristics. The process is attractive to researchers because it reduces the investment in instrumentation and setup. Here we describe a procedure that eliminates the transfer step of western blotting and allows for antigen detection directly within the polyacrylamide gel, thus minimizing the investment necessary for setting up western blotting.

Coated microwell plate-based affinity purification of antigens.

Antibody-based affinity capture of antigens is widely used in the isolation of antigens from complex mixtures. Antibody and the corresponding antigen are allowed to interact with each other to form immunocomplexes which are then typically captured by protein A or protein G immobilized on beaded support. Antigen capture performed using this method generally requires multiple centrifugation steps and careful pipetting to avoid loss of the bead-bound complexes. This traditional procedure is tedious and not easily reproducible, especially when working with multiple samples. To address these issues we have demonstrated that antigens can be captured with protein A/G, protein G, and high binding-capacity streptavidin 96-well strip-coated plates. The coated plate method of antigen purification is reproducible, within the same experiment and between experiments, due to the uniform binding capacity of the plates and wells. Here we report the use of coated microwell plates for antigen capture and for protein-protein interaction studies with the well-characterized BIR2-SMAC, transferrin receptor/ transferrin, and other systems.