This document summarizes the technique of immunoprecipitation. It describes immunoprecipitation as a method to precipitate protein antigens out of solution using an immobilized specific antibody. The document outlines different applications of immunoprecipitation including measuring protein molecular weight, detecting post-translational modifications, and analyzing protein-protein interactions. It also describes different types of immunoprecipitation techniques such as co-immunoprecipitation, chromatin immunoprecipitation, and RNA immunoprecipitation.

1. Immunoprecipitation
Aishwarya Babu
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2. Immunoprecipitation
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• Technique of precipitating protein antigens out of a
solution using a specific antibody which is
immobilized to a solid support.
• Widely used method for protein isolation from
complex samples such as cell lysates, serum and
tissue homogenates.
• Applications:
-to measure the molecular weight of the given
protein
-to determine post translational modifications
-to analyze the expression level of a protein of
interest and
-in the study of protein-protein as well as protein-
nucleic acid interactions.

3. Methods
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 Direct method/Pre-immobilized
antibody approach
-The preferred method when the
target protein is abundant.
-Requires less primary Ab.
 Indirect method/Free antibody
approach
-The preferred method when the
Ab has poor affinity for the target or
when the target protein is in low
abundance

4. Magnetic beads vs. agarose resin for
immunoprecipitation
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 Magnetic beads provide the
best balance of capacity/yield,
reproducibility, purity, and cost
savings for routine small-scale
isolation of specific proteins
and protein complexes.
 Agarose is most appropriate
for large-scale IP reactions
when sufficient antibody is
plentiful at a low cost and
where the goal is to purify a
sufficient amount of target
protein for multiple
downstream assays.

5. Types of immunoprecipitation
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Co-immunoprecipitation
 Used to analyze protein–protein interactions.
 Main purpose of Co-IP is the identification of
interaction partners (other proteins, ligands, co-
factors, or signaling molecules) to the protein of
interest.

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Chromatin Immunoprecipitation (ChIP)
 Used to investigate regions of the genome
associated with a target DNA-binding protein,
or to identify specific proteins associated with
a particular region of the genome.
 Main steps: Crosslinking, cell lysis, chromatin
preparation, IP, reverse crosslinking, DNA
purification and quantitation.

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RNA Immunoprecipitation (RIP)
• Targets RNA-binding proteins (ribonucleoproteins).
• Performed using an antibody that targets a specific
RNA-binding protein.
• RNA-protein complexes are separated by RNA
extraction.

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Tagged protein IP
 Proteins can be tagged with an epitope to which a high affinity
antibody is available and expressed in the cell of interest.
 Tags can be either short peptide sequences or fluorescent proteins,
including: FLAG, c-Myc, Hemagglutinin (HA), Green fluorescent
protein (GFP).
Limitation:
 Overexpressed tagged protein is immunoprecipitated
 Tagging the protein may interfere with protein function.

9. Antibody immobilization
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1. Antibody-Binding
proteins
2. Covalent antibody
immobilization
3. Crosslinking antibody
immobiliaztion

10. IP Buffers
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Lysis buffers
 Stabilizes native protein conformation,
 Inhibits enzymatic activity,
 Maximizes the release of proteins from the cells or tissue.
 Non-denaturing buffers: NP-40 or Triton X-100
 Denaturing buffers: radio-immunoprecipitation assay (RIPA)
 Contains NaCl and Tris-HCl and have a slightly basic pH (7.4 to 8).
 Proteasomal inhibitors: PMSF, aprotinin and leupeptin

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Wash buffers
 Multiple washes with simple wash buffers, such as PBS either alone
or with low detergent concentrations can be used to remove
contaminants.
Elution buffers
 Elution directly in reducing SDS-PAGE sample buffer.
 Non-denaturing elution buffer for protein purification: 0.1 M glycine at
pH 2.5 to 3.
 Low pH condition dissociates antibody-antigen interactions, as well
as the antibody-Protein A/G interaction.

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