Psychopharmacology Lab Wet Assays
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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.

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

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    Psychopharmacology Lab Wet Assays Psychopharmacology Lab Wet Assays Presentation Transcript

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