Transcript Typical IP Protocol

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
CNBr

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
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
agarose
G
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, posttranslational events, etc.
Immunoblotting
•
•
aka Western Blotting
use Ab to detect protein after
electrophoresis
1.
2.
3.
4.
5.
Protein electrophoresis
Transfer proteins to membrane
Incubate membrane with antibody
Extensive washing
Detect bound antibody
- radioactive 2nd Ab or protein A
- ELISA
Enzyme-Linked ImmunoSorbent Assay
Detection by Chemiluminescence
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
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
Immunoassay Uses
• antigen detection, characterization, (quantification?)
• antibody detection, characterization, (quantification?)