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?