Summary of Available Wet Assays

Bradley Wetzell, Psychopharmacology Laboratory

Summary of Available Wet Summary of Available Wet AssaysAssays• Western Blotting (Protein Immunoblot)

• Fluorescence Microscopy• High-Performance Liquid

Chromatography (HPLC)

• Enzyme-Linked Immunosorbent Assay (ELISA)

• qPCR

Western BlottingWestern 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)(Protein Immunoblot)

Western BlottingWestern Blotting(Protein Immunoblot)(Protein Immunoblot)

Western BlottingWestern 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)(Protein Immunoblot)

Fluorescence MicroscopyFluorescence 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

Hippocampus

Cerebellum

Fluorescence MicroscopyFluorescence Microscopy

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 MicroscopyFluorescence Microscopy

High-Performance Liquid High-Performance Liquid ChromatographyChromatography

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 HCl04 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)(HPLC)

High-Performance Liquid High-Performance Liquid ChromatographyChromatography(HPLC)(HPLC)

High-Performance Liquid High-Performance Liquid ChromatographyChromatography

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)(HPLC)

Enzyme-Linked Immunosorbent Enzyme-Linked Immunosorbent AssayAssayWhat 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)(ELISA)

Enzyme-Linked Immunosorbent Enzyme-Linked Immunosorbent AssayAssay(ELISA)(ELISA)

Enzyme-Linked Immunosorbent Enzyme-Linked Immunosorbent AssayAssay

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)(ELISA)

Reverse-Transcription Polymerase Reverse-Transcription Polymerase Reaction TestingReaction 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)(qPCR)

Reverse-Transcription Polymerase Reverse-Transcription Polymerase Reaction TestingReaction Testing(qPCR)(qPCR)

Reverse-Transcription Polymerase Reverse-Transcription Polymerase Reaction TestingReaction 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)(qPCR)

Final ThoughtsFinal ThoughtsThese 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