Summary of Available Wet Assays Bradley Wetzell, Psychopharmacology Laboratory
Summary of Available Wet Assays <ul><li>Western Blotting  (Protein Immunoblot) </li></ul><ul><li>Fluorescence Microscopy <...
Western Blotting <ul><li>What does it do? </li></ul><ul><ul><li>Detects the presence of specific proteins in tissue </li><...
Western Blotting <ul><li>(Protein Immunoblot) </li></ul>
Western Blotting <ul><li>Advantages </li></ul><ul><ul><li>Sensitive test </li></ul></ul><ul><ul><li>Nice, clean easily int...
Fluorescence Microscopy <ul><li>What does it do? </li></ul><ul><ul><li>Detects the presence of specific proteins  in-situ ...
Fluorescence Microscopy Hippocampus Cerebellum
<ul><li>Advantages </li></ul><ul><ul><li>View proteins  in-situ </li></ul></ul><ul><ul><li>Simple and quick </li></ul></ul...
High-Performance Liquid Chromatography <ul><li>What does it do? </li></ul><ul><ul><li>Detects the presence of smaller mole...
High-Performance Liquid Chromatography <ul><li>(HPLC) </li></ul>
High-Performance Liquid Chromatography <ul><li>Advantages </li></ul><ul><ul><li>Molecular-level detection of smaller compo...
Enzyme-Linked Immunosorbent Assay <ul><li>What does it do? </li></ul><ul><ul><li>Detects the presence of specific proteins...
Enzyme-Linked Immunosorbent Assay <ul><li>(ELISA) </li></ul>
Enzyme-Linked Immunosorbent Assay <ul><li>Advantages </li></ul><ul><ul><li>Very quick and easy test (96 samples at a time)...
Reverse-Transcription Polymerase Reaction Testing <ul><li>What does it do? </li></ul><ul><ul><li>Detects the presence of m...
Reverse-Transcription Polymerase Reaction Testing <ul><li>(qPCR) </li></ul>
Reverse-Transcription Polymerase Reaction Testing <ul><li>Advantages </li></ul><ul><ul><li>Determine gene transcription so...
Final Thoughts <ul><li>These procedures are generalizable </li></ul><ul><ul><li>Many of these tests use similar procedures...
 
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Psychopharmacology Lab Wet Assays

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A brief overview to familiarize new lab members with available biological assays in the lab.

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Psychopharmacology Lab Wet Assays

  1. 1. Summary of Available Wet Assays Bradley Wetzell, Psychopharmacology Laboratory
  2. 2. Summary of Available Wet Assays <ul><li>Western Blotting (Protein Immunoblot) </li></ul><ul><li>Fluorescence Microscopy </li></ul><ul><li>High-Performance Liquid Chromatography (HPLC) </li></ul><ul><li>Enzyme-Linked Immunosorbent Assay (ELISA) </li></ul><ul><li>qPCR </li></ul>
  3. 3. Western Blotting <ul><li>What does it do? </li></ul><ul><ul><li>Detects the presence of specific proteins in tissue </li></ul></ul><ul><li>How does it do it? </li></ul><ul><ul><li>Tissue is homogenized in buffer to lyse cells and free bound proteins </li></ul></ul><ul><ul><li>Lysate is transferred to gel plates and proteins are separated according to size by electrophoresis </li></ul></ul><ul><ul><li>‘ Tagged’ antibodies that bind to protein of interest are added to the gel and allowed to bind </li></ul></ul><ul><ul><li>Tags detected with fluorescence detector </li></ul></ul><ul><ul><li>Fluorescence = protein </li></ul></ul><ul><li>(Protein Immunoblot) </li></ul>
  4. 4. Western Blotting <ul><li>(Protein Immunoblot) </li></ul>
  5. 5. Western Blotting <ul><li>Advantages </li></ul><ul><ul><li>Sensitive test </li></ul></ul><ul><ul><li>Nice, clean easily interpretable output </li></ul></ul><ul><ul><li>Thousands of commercially available antibodies </li></ul></ul><ul><ul><li>Tests denatured proteins, so changes in functional states can be detected (i.e., receptor phosphorylation, acetylation, methylation, etc.) </li></ul></ul><ul><li>Disadvantages </li></ul><ul><ul><li>Not quick, slightly labor-intensive </li></ul></ul><ul><ul><li>Tissue must be homogenized </li></ul></ul><ul><li>(Protein Immunoblot) </li></ul>
  6. 6. Fluorescence Microscopy <ul><li>What does it do? </li></ul><ul><ul><li>Detects the presence of specific proteins in-situ (also sometimes called ‘ in-situ hybridization’) </li></ul></ul><ul><li>How does it do it? </li></ul><ul><ul><li>Tissue is ‘fixed’ to cross-link proteins </li></ul></ul><ul><ul><li>Fixed tissue is cryosectioned and mounted to slides </li></ul></ul><ul><ul><li>Slides are washed with tagged antibodies that bind to protein of interest </li></ul></ul><ul><ul><li>Fluorescence microscope used to detect tags </li></ul></ul><ul><ul><li>Presence and density of tags=protein </li></ul></ul><ul><ul><li>Detection by software </li></ul></ul>
  7. 7. Fluorescence Microscopy Hippocampus Cerebellum
  8. 8. <ul><li>Advantages </li></ul><ul><ul><li>View proteins in-situ </li></ul></ul><ul><ul><li>Simple and quick </li></ul></ul><ul><ul><li>Specific detection </li></ul></ul><ul><ul><li>Nice, easily interpretable output </li></ul></ul><ul><ul><li>Analysis is software-driven (in our lab) </li></ul></ul><ul><li>Disadvantages </li></ul><ul><ul><li>Tissue must be fixed (transcardial perfusion) </li></ul></ul><ul><ul><li>Rapid bleaching </li></ul></ul><ul><ul><li>Possibility of phototoxicity </li></ul></ul><ul><ul><li>Out of focus artifacts can degrade image </li></ul></ul>Fluorescence Microscopy
  9. 9. High-Performance Liquid Chromatography <ul><li>What does it do? </li></ul><ul><ul><li>Detects the presence of smaller molecules in tissue or serum (i.e., neurotransmitters, drugs, metabolites) </li></ul></ul><ul><li>How does it do it? </li></ul><ul><ul><li>Tissue is homogenized in HCl0 4 to lyse cells and vesicles and free bound molecules (serum or plasma are tested with weaker acids) </li></ul></ul><ul><ul><li>Lysate is forced through a column ‘filter’ at high pressure </li></ul></ul><ul><ul><li>Separates molecules by size and those of similar size exit the column at the same time </li></ul></ul><ul><ul><li>Pass through an electrical field where they are oxidized </li></ul></ul><ul><ul><li>Detects time and intensity of oxidizing reactions </li></ul></ul><ul><li>(HPLC) </li></ul>
  10. 10. High-Performance Liquid Chromatography <ul><li>(HPLC) </li></ul>
  11. 11. High-Performance Liquid Chromatography <ul><li>Advantages </li></ul><ul><ul><li>Molecular-level detection of smaller compounds </li></ul></ul><ul><ul><li>Fairly quick analysis (lengthy prep) </li></ul></ul><ul><ul><li>Because of high pressure, compounds are well separated and easy to read </li></ul></ul><ul><li>Disadvantages </li></ul><ul><ul><li>Operation can be complex (but we’re working to make it more ‘fool-proof’ for compounds we commonly test </li></ul></ul><ul><ul><li>Some compounds can be difficult or impossible to detect (i.e., opiates and rapidly absorbed molecules) </li></ul></ul><ul><ul><li>Need to know theoretical concentrations of compound of interest to prepare standards </li></ul></ul><ul><li>(HPLC) </li></ul>
  12. 12. Enzyme-Linked Immunosorbent Assay <ul><li>What does it do? </li></ul><ul><ul><li>Detects the presence of specific proteins in tissue and serum. Can also detect antibodies, hormones, drug compounds and metabolites </li></ul></ul><ul><li>How does it do it? </li></ul><ul><ul><li>Tissue is homogenized in buffer to lyse cells and free bound proteins </li></ul></ul><ul><ul><li>96 well plate is coated with antibodies that bind to protein of interest </li></ul></ul><ul><ul><li>Lysate is added to well, if protein is present it will bind </li></ul></ul><ul><ul><li>Additional ‘tagged’ antibodies are added that also bind to bound protein </li></ul></ul><ul><ul><li>Tag = enzyme, substrate is added, enzyme produces visible product </li></ul></ul><ul><ul><li>Visible product = protein </li></ul></ul><ul><li>(ELISA) </li></ul>
  13. 13. Enzyme-Linked Immunosorbent Assay <ul><li>(ELISA) </li></ul>
  14. 14. Enzyme-Linked Immunosorbent Assay <ul><li>Advantages </li></ul><ul><ul><li>Very quick and easy test (96 samples at a time) </li></ul></ul><ul><ul><li>Wide variety of commercially available kits to detect specific proteins </li></ul></ul><ul><ul><li>Detects only proteins that are properly folded (this could be useful for research purposes) </li></ul></ul><ul><li>Disadvantages </li></ul><ul><ul><li>Only uses monoclonal antibodies (more expensive) </li></ul></ul><ul><ul><li>Can underestimate some protein levels (because it can’t detect denatured proteins) </li></ul></ul><ul><ul><li>Enzyme:Substrate reaction is short, must be read and recorded quickly </li></ul></ul><ul><li>(ELISA) </li></ul>
  15. 15. Reverse-Transcription Polymerase Reaction Testing <ul><li>What does it do? </li></ul><ul><ul><li>Detects the presence of mRNA sequences in tissue </li></ul></ul><ul><li>How does it do it? </li></ul><ul><ul><li>Tissue is homogenized in buffer to lyse cells and free nucleotides </li></ul></ul><ul><ul><li>Enzyme (thermostable DNA polymerase) and ‘tagged’ oligonucleotide sequences are added to lysate and bind to any available mRNA sequences of interest </li></ul></ul><ul><ul><li>Tagged sample is thermocycled to amplify the sequences </li></ul></ul><ul><ul><li>Fluorescence detectors look for amplified tag </li></ul></ul><ul><ul><li>Fluorescence = mRNA of interest </li></ul></ul><ul><ul><li>Number of cycles to detection determines density </li></ul></ul><ul><li>(qPCR) </li></ul>
  16. 16. Reverse-Transcription Polymerase Reaction Testing <ul><li>(qPCR) </li></ul>
  17. 17. Reverse-Transcription Polymerase Reaction Testing <ul><li>Advantages </li></ul><ul><ul><li>Determine gene transcription sooner </li></ul></ul><ul><ul><li>Can couple with protein analyses to determine latency between transcription and product </li></ul></ul><ul><ul><li>Most sensitive and accurate mRNA detection and quantitation technique available today </li></ul></ul><ul><li>Disadvantages </li></ul><ul><ul><li>Requires careful setup and prep to insure against unwanted artifacts </li></ul></ul><ul><ul><li>RNA is fragile, template modification/degradation must be guarded against </li></ul></ul><ul><ul><li>Possibility of unknown inhibitory substances in sample </li></ul></ul><ul><li>(qPCR) </li></ul>
  18. 18. Final Thoughts <ul><li>These procedures are generalizable </li></ul><ul><ul><li>Many of these tests use similar procedures with minor tweaks </li></ul></ul><ul><ul><li>If you learn a couple of them, you can easily adapt the skills to other tests </li></ul></ul><ul><ul><li>There are many more tests out there that we can likely do… explore options, read the available literature </li></ul></ul>

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