Autoradiography:

See Karp, chapter 18, section 18.4, pp. 730-731 Autoradiography is used to detect and to localize cell components (e.g., proteins; mRNA) based on their incorporation of radioactive precursor molecules. An alternative approach covalently links a radioactive tag to pre-existing macromolecules. Both techniques generate isotopically labeled macromolecules that can easily be detected by autoradiography against a negative background of unlabeled molecules and structures. The most commonly used isotopes for labeling biological material are: 14C amino acids; 3H amino acids; or 35S amino acids for labeling protein synthesis. 3H thymidine; or 32P - thymidine for labeling DNA synthesis 3H uridine or 32P uridine for labeling RNA synthesis Pre-existing proteins and other macromolecules with free NH2 groups can also be labeled by the Iodogen Reaction, which enzymatically links 125 Iodine or 131 Iodine to free amino groups. (carbohydrate and lipid synthesis can also be isotopically labeled by various means) Experimental protocol: 1. Expose live cells or tissues in culture, or inject live animals, with radioisotope precursor of choice. (e.g., 14C leucine to label proteins being synthesized) 2. After desired labeling interval (either continuous or pulse-chase), fix and section cell or tissue specimen and place on microscope slide. 3. Cover specimen with a photographic emulsion containing AgNO3 or cover with X-ray film. 4. Let specimen develop (beta emissions from isotope decay reduce Ag+ to Ago, creating a black spot or track of emissions). The number and intensity of the spots are proportional to the number of macromolecules that have incorporated the isotope. 5. Examine (usually by light or electron microscopy) specimen for black spots revealing the location of the protein, DNA, or RNA that was synthesized during the exposure to the labeled precursor. Note: autoradiography can also be performed on blots of proteins (western blots) or nucleic acids (southern and northern blots) after they have been resolved by electrophoresis.