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Flow cytometry gel electrophoresis immunoprecipitation (IP) immunoblotting microscopy immunofluorescence (IFA) electron microscopy ELISA Analytical Techniques.

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Presentation on theme: "Flow cytometry gel electrophoresis immunoprecipitation (IP) immunoblotting microscopy immunofluorescence (IFA) electron microscopy ELISA Analytical Techniques."— Presentation transcript:

1 flow cytometry gel electrophoresis immunoprecipitation (IP) immunoblotting microscopy immunofluorescence (IFA) electron microscopy ELISA Analytical Techniques Utilizing Antibodies

2 Immunofluorescence Used 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)

3 IFA Protocol 1.Prepare cells or tissue. 2.Incubate 1 o antibody. 3.Wash. 4.Incubate 2 o 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

4 epifluorescence bright field bright image against dark background corresponds to location of antigen

5 + 0.1% TX-100 Detergent Permeabilization

6 EtBr stains DNA and RNA DAPI stains only DNA Counter Staining with Fluorescent Dyes

7 Dual-Labeling Experiments determine extent of co-localization use 1 o antibodies from different species and 2 o antibodies labeled with different fluorochromes label 1 o antibodies with different fluorochromes

8 Immuno-Electron Microscopy prepare samples optimize fixation conditions use resins that polymerize at RT float grids on drops (1 o and 2 o abs, washes) surface of section accessible to antibodies ( ± 'etching') 2 o -Ab conjugated with colloidal gold size ranges from 5-15 nm enzyme linked (electron dense precipitate)

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10 Ultrastructure vs. Labeling fixation conditions preserving ultrastructure lead to loss of labeling cryo-electron microscopy special microtome and stage

11 Accessibility Problems ultrasmall gold (<1 nm) + silver enhancement ±pre-embedding

12 Use same antigen with different antibodies Quantify by serial dilutions Characterizing Antibodies IFA

13 bind antigen to 96-well microplate (or membrane) neg. (and pos.) controls purity? incubate with 1 o and 2 o antibodies use soluble chromogenic substrates in 96-well plates quantify Ab or Ag Conventional ELISA

14 measure absorbance with ELISA microplate reader

15 ELISA Variations radio-immunosorbent assay (RIA)

16 Generic Immunoassay Procedure form antibody-antigen complex detect Ag-Ab complex labeled anti-antibody (2 o Ab) labeled protein A or G directly label 1 o Ab Directvs.Indirect less steps less bkg (?) dual label convenience amplification (?) Fluorochromes fluorescein rhodamine Enzyme Crosslinking AP HRP Radiolabeling iodination metabolically (mAbs) Biotinylation

17 Biotin-Avidin Detection Systems label 1 o - or 2 o -Ab with biotin detect with avidin labeled with marker high affinity may increase sensitivity more steps

18 TECHNIQUE GENERAL 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?


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