1. Antibodies Analytical Techniques Utilizing Antibodies: flow cytometry
gel electrophoresis
immunoprecipitation (IP)
immunoblotting
microscopy
immunofluorescence (IFA)
electron microscopy
ELISA
CNBr 
antibodies bind proteins with high specificity and affinity
affinity chromatography
analytical techniques

2. Immunoprecipitation
affinity purification based on isolation of Ag-Ab complexes
analyze by gel electrophoresis
initially based on centrifugation of large supramolecular complexes
[high] and equal amounts
isolation of Ag-Ab complexes
fixed S. aureus
protein A-agarose
protein G-agarose
Bacterial proteins that bind IgG (Fc):
protein A (Staphylococcus aureus)
protein G (Streptococcus)
binds more species and subclasses

3. Typical IP Protocol G agarose 1. Solubilize antigen
usually non-denaturing
SDS + excess of TX100
2. Mix extract and Ab
3. Add protein G-agarose, etc
4. Extensively wash
5. Elute with sample buffer
6. SDS-PAGE
7. Detection
protein stain
radioactivity
agarose G

4. Radiolabeling of Proteins
carried out before IP
metabolic (amino acids or other precursors + cells)
chemically (eg., iodination)
IP and SDS-PAGE
detect by autoradiography or fluorography following electrophoresis
also provides information about synthesis, post-translational events, etc.

5. Immunoblotting aka Western Blotting
use Ab to detect protein after electrophoresis
1. Protein electrophoresis
2. Transfer proteins to membrane
3. Incubate membrane with antibody
4. Extensive washing
5. Detect bound antibody
- radioactive 2nd Ab or protein A
- ELISA

6. Enzyme-Linked ImmunoSorbent Assay

7. Detection by Chemiluminescence

8. Western Blot vs Immunoprecipitation
Experimental Design
eg., synthesis (IP)
Ag concentration
IP better for low abundance proteins
Ag solubility
Western for insoluble proteins
Ab recognition
conformational dependent epitopes
4o structure

9. Combined Immunoprecipitation and Immunoblotting
carry out IP and electrophoresis
transfer to membrane
use same or different Ab against Ag in blotting
need ‘no extract’ control

10. Immunoassay Uses
antigen detection, characterization, (quantification?)
antibody detection, characterization, (quantification?)