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Analytical Techniques Utilizing Antibodies. flow cytometry gel electrophoresis immunoprecipitation (IP) immunoblotting microscopy immunofluorescence (IFA) electron microscopy ELISA. Immunofluorescence. Used to : localize antigens to specific cells or subcellular structures - PowerPoint PPT Presentation
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• flow cytometry• gel electrophoresis• immunoprecipitation (IP)• immunoblotting
• microscopy• immunofluorescence (IFA)• electron microscopy
• ELISA
Analytical Techniques Utilizing Antibodies
ImmunofluorescenceUsed to:• localize antigens to specific cells or
subcellular structures• detect and quantify antibody
General Procedure:• incubate cells or tissue with antibody• detect Ag-Ab complex with conjugated
secondary antibody• fluorescence (examine under UV
illumination)• enzyme (substrate forms precipitate)
IFA Protocol
1. Prepare cells or tissue.
2. Incubate 1o antibody.
3. Wash.4. Incubate 2o
antibody.5. Wash.6. View under UV
illumination.
Preparation of Cells• section and mount tissues• grow adherent cells on micro-
scope slides or cover slips• affix suspension cells on
microscope slides• carry out incubations in
suspension• ± fixation • organic solvents• paraformaldehyde• <0.1% glutaraldehyde
• ± permeabilization (eg., detergents)
• surface labeling of unfixed cells
epifluorescence bright field
•bright image against dark background•corresponds to location of antigen
+ 0.1% TX-100
Detergent Permeabilization
EtBr stains DNA and RNA
DAPI stains only DNA
Counter Staining with Fluorescent
Dyes
Dual-Labeling Experiments
•determine extent of co-localization•use 1o antibodies from different species and 2o antibodies labeled with different fluorochromes
•label 1o antibodies with different fluorochromes
Immuno-Electron Microscopy
• prepare samples• optimize fixation conditions• use resins that polymerize at RT
• ‘float’ grids on drops (1o and 2o abs, washes)• surface of section accessible to
antibodies (± 'etching')• 2o-Ab conjugated with colloidal gold• size ranges from 5-15 nm• enzyme linked (electron dense precipitate)
Ultrastructure vs. Labeling
• fixation conditions preserving ultrastructure lead to loss of labeling
• cryo-electron microscopy• special microtome and
stage
AccessibilityProblems
• ultrasmall gold (<1 nm)• + silver
enhancement• ±pre-embedding
• Use same antigen with different antibodies• Quantify by serial dilutions
Characterizing Antibodies
IFA
• bind antigen to 96-well microplate (or membrane)• neg. (and pos.) controls• purity?
• incubate with 1o and 2o antibodies• use soluble chromogenic
substrates in 96-well plates• quantify Ab or Ag
Conventional ELISA
• measure absorbance with ELISA microplate reader
ELISA Variations
• radio-immunosorbent assay (RIA)
Generic Immunoassay Procedure•form antibody-antigen complex•detect Ag-Ab complex•labeled anti-antibody (2o Ab)•labeled protein A or G•directly label 1o Ab
Direct vs.
Indirect
less stepsless bkg (?)dual label
convenienceamplification (?)
Fluorochromes•fluorescein• rhodamineEnzyme
Crosslinking•AP•HRP Radiolabeling• iodination•metabolically
(mAbs)Biotinylation
Biotin-Avidin Detection Systems
•label 1o- or 2o-Ab with biotin
•detect with avidin labeled with marker
•high affinity may increase sensitivity
•more steps
TECHNIQUEGENERAL PROCEDURE
TYPICAL APPLICATION
ELISA
adsorb Ag to solid support
incubate with Ab detect bound Ab
quantify Ab quantify Ag process large # of
samples
BLOTTING
SDS-PAGE and transfer
incubate with Ab detect bound Ab
identify subunit MW quantify Ag quantify Ab?
IFA fix cells on slide incubate with Ab detect bound Ab
subcellular localization quantify Ab quantify Ag?