will not be carried toward the anode, since transfer takes place on the basis of charge. In these cases it is best to include SDs in the transfer buffer. Alternatively, the transfer sandwich can be assem bled with membranes on both sides of the gel CHOOSING A DETECTION STRATEGY OVERVIEW OF DETECTION SYSTEMS Detection systems range from the simplest colorimetric systems for use on the benchtop to complex instrument-based systems Table 13. 1). The simplest is radioactive detection: a secondary reagent is labeled with a radioactive isotope, usually the low- energy gamma-emitter iodine-125. After the blot is incubated with the primary antibody, the labeled secondary rotein A, but it can be a secondary antibody) is applied, the blot Table 13.1 Comparison of Detection Methods Method Features Limitations Radioactive Can quantitate Use of radioactive lpg material can be densitometry; difficult and expensive obe bl requ no enzymatic licensing radiat training Colorimetric Easy to perform; Relatively 200pg hard cop entitiy results directly on blot. for facilities and equipment Chemiluminescent Highly sensitive; Requires careful l pg(luminol) an quantitate optimization using film 0.1 pg(acridan) metry, Fluorescent Good linear ange for stringent data stored membrane digit d possible but difficult Western blotting 375will not be carried toward the anode, since transfer takes place on the basis of charge. In these cases it is best to include SDS in the transfer buffer. Alternatively, the transfer sandwich can be assem￾bled with membranes on both sides of the gel. CHOOSING A DETECTION STRATEGY: OVERVIEW OF DETECTION SYSTEMS Detection systems range from the simplest colorimetric systems for use on the benchtop to complex instrument-based systems (Table 13.1). The simplest is radioactive detection: a secondary reagent is labeled with a radioactive isotope, usually the low￾energy gamma-emitter iodine-125.After the blot is incubated with the primary antibody, the labeled secondary reagent (usually Protein A, but it can be a secondary antibody) is applied, the blot Western Blotting 375 Table 13.1 Comparison of Detection Methods Method Features Limitations Sensitivity Radioactive Can quantitate Use of radioactive 1 pg through film material can be densitometry; difficult and can strip and expensive; reprobe blots; requires no enzymatic licensing and development radiation safety step training Colorimetric Easy to perform; Relatively 200 pg hard copy insensitive results directly on blot; minimal requirements for facilities and equipment Chemiluminescent Highly sensitive; Requires careful 1 pg (luminol) can quantitate optimization using film 0.1 pg (acridan) densitometry; can strip and reprobe Fluorescent Good linear Equipment 1 pg range for expensive; quantitation; stringent data stored membrane digitally requirements; stripping and reprobing possible but difficult