flow cytometry gel electrophoresis immunoprecipitation (ip) immunoblotting microscopy

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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 cytometrygel electrophoresisimmunoprecipitation (IP)immunoblottingmicroscopyimmunofluorescence (IFA)electron microscopyELISAAnalytical Techniques Utilizing Antibodies

  • ImmunofluorescenceUsed to:localize antigens to specific cells or subcellular structuresdetect and quantify antibody

    General Procedure:incubate cells or tissue with antibodydetect Ag-Ab complex with conjugated secondary antibodyfluorescence (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 Cellssection and mount tissuesgrow adherent cells on micro-scope slides or cover slipsaffix suspension cells on microscope slidescarry out incubations in suspension fixation organic solventsparaformaldehyde

  • epifluorescencebright fieldbright image against dark backgroundcorresponds to location of antigen

  • + 0.1% TX-100Detergent Permeabilization

  • EtBr stains DNA and RNACounter Staining with Fluorescent Dyes

  • Dual-Labeling Experimentsdetermine extent of co-localizationuse 1o antibodies from different species and 2o antibodies labeled with different fluorochromeslabel 1o antibodies with different fluorochromes

  • Immuno-Electron Microscopyprepare samplesoptimize fixation conditionsuse resins that polymerize at RTfloat grids on drops (1o and 2o abs, washes)surface of section accessible to antibodies ( 'etching')2o-Ab conjugated with colloidal goldsize ranges from 5-15 nmenzyme linked (electron dense precipitate)

  • Ultrastructure vs. Labelingfixation conditions preserving ultrastructure lead to loss of labeling

    cryo-electron microscopyspecial microtome and stage

  • AccessibilityProblemsultrasmall gold (
  • Use same antigen with different antibodiesQuantify by serial dilutionsCharacterizing Antibodies

  • bind antigen to 96-well microplate (or membrane)neg. (and pos.) controlspurity?incubate with 1o and 2o antibodiesuse soluble chromogenic substrates in 96-well platesquantify Ab or AgConventional ELISA

  • measure absorbance with ELISA microplate reader

  • ELISA Variationsradio-immunosorbent assay (RIA)

  • Generic Immunoassay Procedureform antibody-antigen complexdetect Ag-Ab complexlabeled anti-antibody (2o Ab)labeled protein A or Gdirectly label 1o AbFluorochromesfluoresceinrhodamineEnzyme CrosslinkingAPHRP Radiolabelingiodinationmetabolically (mAbs)Biotinylation

    Directvs.Indirectless stepsless bkg (?)dual labelconvenienceamplification (?)

  • Biotin-Avidin Detection Systemslabel 1o- or 2o-Ab with biotindetect with avidin labeled with markerhigh affinity may increase sensitivitymore steps

  • TECHNIQUEGENERAL PROCEDURETYPICAL APPLICATIONELISAadsorb Ag to solid supportincubate with Abdetect bound Abquantify Abquantify Agprocess large # of samplesBLOTTINGSDS-PAGE and transferincubate with Abdetect bound Abidentify subunit MWquantify Agquantify Ab?IFAfix cells on slideincubate with Abdetect bound Absubcellular localizationquantify Abquantify Ag?

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