digital raw data from fluorescence scanners (and storag phosphor scanners for radioactive detection) is inherently quan titative. The linear range of film-based systems (limited by the response of the film)is a little better than one order of magnitude hile the manufacturers of fluorescent scanners claim something closer to two orders of magnitude There are several cautions to bear in mind when considering protein blot quantitation Standards(known amounts of purified target protein-not to be confused with molecular weight stan dards)must be run on every blot, since even with the most con- stent technique there can be blot-to-blot variation. The standard should be loaded on the gel, electrophoresed, and transferred in exactly the same way your samples are The determination of quantity can only be made within the ange of standards on the blot: extrapolation beyond the actual standard values is not valid. This together with the limited linear range means that several dilutions of the unknown sample usually must be run on the same blot given all the lanes of standards and sample dilutions, the amount of quantitative data that can be extracted from a single blot is somewhat limited. Protein blot uantitation can be useful, but it is not a substitute for techniques such as elisa or ria Antibody requirements Typically the choice of available primary antibodies is not as vide as that of the other elements of the detection system. Primary antibodies can be obtained from commercial suppliers, non-profit repositories, and even other researchers. Tracking down a primary antibody can be time-consuming, but publications such as Lin- scottsDirectory(linscott,1999,andhttp://www.linscottsdirec tory. com/index2hmD,the“ Antibody Resource Page”(htp∥ www.antibodyresource.com),theUsenetnewsgroup"methods and Reagents"(bionet. molbio. methds-reagnts ), and Stefan Dubel's recombinantantibodypage(www.mgen.uni-heidelberg.de/sd/ SdscFvsite.htmlandwww.antibody.comcanhelp If no antibodies against your target protein exist, your only options are to raise the antibody yourself or to have someone do it. Companies such as Berkeley Antibody Company, Genosys, Rockland, and Zymed(among many others) can do his kind of work on a contract basis. Whichever route you choose, this is a time-consuming and potentially expensive undertaking 378 Riisdigital raw data from fluorescence scanners (and storage￾phosphor scanners for radioactive detection) is inherently quan￾titative. The linear range of film-based systems (limited by the response of the film) is a little better than one order of magnitude, while the manufacturers of fluorescent scanners claim something closer to two orders of magnitude. There are several cautions to bear in mind when considering protein blot quantitation. Standards (known amounts of purified target protein—not to be confused with molecular weight stan￾dards) must be run on every blot, since even with the most con￾sistent technique there can be blot-to-blot variation. The standard should be loaded on the gel, electrophoresed, and transferred in exactly the same way your samples are. The determination of quantity can only be made within the range of standards on the blot: extrapolation beyond the actual standard values is not valid. This together with the limited linear range means that several dilutions of the unknown sample usually must be run on the same blot. Given all the lanes of standards and sample dilutions, the amount of quantitative data that can be extracted from a single blot is somewhat limited. Protein blot quantitation can be useful, but it is not a substitute for techniques such as ELISA or RIA. Antibody Requirements Typically the choice of available primary antibodies is not as wide as that of the other elements of the detection system. Primary antibodies can be obtained from commercial suppliers, non-profit repositories, and even other researchers. Tracking down a primary antibody can be time-consuming, but publications such as Lin￾scott’s Directory (Linscott, 1999, and http://www.linscottsdirec￾tory.com/index2.html), the “Antibody Resource Page” (http:// www.antibodyresource.com), the Usenet newsgroup “Methods and Reagents” (bionet.molbio.methds-reagnts), and Stefan Dubel’s recombinant antibody page (www.mgen.uni-heidelberg.de/SD/ SDscFvSite.html) and www.antibody.com can help. If no antibodies against your target protein exist, your only options are to raise the antibody yourself or to have someone else do it. Companies such as Berkeley Antibody Company, Genosys, Rockland, and Zymed (among many others) can do this kind of work on a contract basis. Whichever route you choose, this is a time-consuming and potentially expensive undertaking. 378 Riis