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Extracting Hormones and DNA

This guide details the extraction processes for hormones and DNA from various biological samples, including blood, yolks, and feces. It explores differences in cell types (nucleated in birds vs. non-nucleated in mammals), methods for isolating steroid hormones from plasma, and procedures for extracting DNA. Key protocols such as diethyl ether extraction, chromatography, and competitive binding assays are discussed. This resource aims to assist researchers in optimizing extraction efficiency and improving assay accuracy.

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Extracting Hormones and DNA

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  1. Extracting Hormones and DNA

  2. Blood • Red blood cells • Birds: nucleated • Mammals: non-nucleated • White blood cells • Plasma • Fluids

  3. Steroid Hormones • Precursor is cholesterol: steroid hormones are fat-soluble, not water-soluble • Types of steroid hormones: • Androgens (e.g., testosterone) • Progestins • Estrogens • Corticoids (e.g., corticosterone)

  4. Extraction of Steroid Hormones from Blood • Separate plasma from RBCs • Diethyl ether extraction method • Diethyl ether separates liquid phase of plasma • Snap-freeze: pour off unfrozen ether with steroid hormones • Dry and reconstitute

  5. Chromatography • To study multiple hormones: you will need to isolate each one from this extract • Run the mixture through layers of celite with varying concentrations of ethyl acetate in isooctane

  6. Extraction from Yolks • Yolk is laid down in layers; maternal steroid concentrations differ in each layer • In nonviable eggs: • Separate yolk from white (usually by freezing) • Homogenize yolk (if measuring total concentration) or sample layers • Extraction protocol similar to blood • Use petroleum ether in addition to diethyl ether: separates out lipids as well

  7. Extraction from Feces • Requires additional steps at the beginning of process (drying, pulverizing) • Additional concerns: metabolizing of steroids

  8. Measuring Extraction Efficiency • In any extraction method, you lose a certain amount of the target material • Add known amount of radioactive hormone before extraction • Measure remaining amount of radioactivity after extraction • Gives you a percentage, and you can correct your estimates of “native” hormone accordingly

  9. Types of Assays • EIA: Enzyme ImmunoAssay • RIA: RadioImmunoAssay • Both work on the principle of competitive binding

  10. Competitive Binding • Sample has “native” hormone • Add to sample either radioactive hormone or alkaline phosphatase bound to hormone • Antibody • Competitive binding: native hormone binds to antibody better than nonnative • Measure remaining amount of nonnative hormone: inversely proportional to native hormone in sample

  11. Setting up an Assay • Randomizing samples

  12. Setting up an Assay

  13. Standard Curve

  14. Standards • Known concentrations of hormone • Used as a sample at beginning, middle, and end of samples

  15. DNA Extraction from Blood • Lysis: break open cells • Proteinase K: breaks up proteins • Phenol/Chloroform: removes proteins and other material • Can also use kits

  16. DNA Extraction from Feces • Shed cells from epithelial lining of intestine • A few difficulties: • Lower-quality DNA • PCR inhibitors in feces, such as bile acids

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