Assays and Techniques Assays • Measurement of hormone levels – Bioassays – Validation • Accuracy • Objective endpoint • Specificity • Precision • Sensitivity Assays • Chemical and Physical Assays • Competitive binding Assays • Immunoassays – RIA • Start here 502 Cytological-Histological • Early studies – Anatomical and descriptive using light microscopy • Gross anatomy – Localization, size, blood supply, innervation – Hypertrophy /atrophy • Immunocytochemistry – Indicator labled antibody localization of hormone Surgical Methods • Ablation and replacement experiments – Removal of candidate tissue and observation of physiological changes – Ex. Berthold and the roosters – Compensatory hypertrophy – “ectomies” Hormone Replacement Therapy • Giving of exogenous hormone to replace lost hormone after theablation of endocrine tissues of glands – Ex menopause/ estrogen/progestin therapy Tissue Extracts/Purification • Crude extracts in early studies – Problems • Purifcation • Ex insulin – Ovine, porcine, bovine, now genetically engineered! Chemical Identification + Synthesis • Simple elemental analysis • Primary amino acid sequence • Secondary, tertiary, and quaternary structure • Modifications – glycosylation Bioassays • Measuring the biological activity of a hormone of hormone extract • Need living tissue that produces a detctable physiological response to the hormone • in vivo or in vitro • Physiological vs. Pharmacological doses • Ex Frog skin/MSH • Epiphysial plates/ TSH Radio-isotope studies • Isotopes of I 125, Ca 45, S 35, P 32, Na 23, C 14, H 3 are incorporated into hormones and then tracked • Can determine: – Metabolism, abundance, half- life, enzymatic rates – Ex. Radio immunoassayRadio-isotope studies • Radioreceptor Assays – Radiolabelled hormones bind to their normal membrane receptors, usually dose study • Enzyme Assays – Enzyme activity ex. Adneylate cyclase activity • Autoradiography – Classic – injection of tracer/to tissue, expose tissue to film, develop film to find tracer localization • Ex steroids and nuclear DNA • Hybridization – Paring of nucleic acid fragments – tracer is attached to a probe (short radioactive DNA or RNA sequence), rest like autoradiography. Electrophysiological Methods • Excitable cells! – Monitoring of transmembrane charges with microelectrodes, detects deloparization and hyperpolarizations Pharmacological Methods • Use of drugs or chemicals to study physiological activity in cells • Ex oubain inhibits the Na/K pump • Inhibitors – Cochicine- inhibits microtubule formation • Actinomyosin –d • Receptor agonist/antagonists Genetic Engineering • Microinjection of nucleic acid that incorporate in to the pronuclei of fertilzed cells • Examples – Insulin producing bacteria – Super salmon • Transgenic speciesQuiz 2 • Explain what parabiosis is and how it would help you determine the source of a