Typical IP Protocol

1
Antibodies

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

• 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

• 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

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?)