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Analytical Techniques

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

2 Immunofluorescence Used to: General Procedure:
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 Preparation of Cells IFA Protocol 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 IFA Protocol 1. Prepare cells or tissue. 2. Incubate 1o antibody. 3. Wash. 4. Incubate 2o antibody. 5. Wash. 6. View under UV illumination.

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

5 Detergent Permeabilization
+ 0.1% TX-100

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

7 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

8 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)


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

13 Conventional ELISA 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

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 (2o Ab) labeled protein A or G directly label 1o Ab Direct vs. 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 1o- or 2o-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 identify subunit MW quantify Ab? IFA fix cells on slide subcellular localization quantify Ag?

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