IMMUNOPRECIPITATION

BY18203
Analytical Techniques in
Biotechnology Seminar

Presented By:
Madhavan Yasasve
Reg. No. 190211012
I Year - M. Tech. (Biotechnology)
Sri Venkateswara College of Engineering
(Autonomous - Affiliated to Anna University),
Sriperumbudur

Submitted to:
Mr. P. K. Praveen Kumar
Associate Professor
Department of Biotechnology,
Sri Venkateswara College of Engineering
(Autonomous - Affiliated to Anna University),
Sriperumbudur
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 CONTENTS

1. INTRODUCTION

2. BASIC OUTLINE OF IMMUNOPRECIPITATION

3. METHODS USED

4. TYPES OF IMMUNOPRECIPITATION

5. FACTORS INFLUENCING IP

6. IMMUNOPRECIPITATION PROTOCOL

7. APPLICATIONS

8. TROUBLESHOOTING
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 INTRODUCTION
• It is a 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

• Immunoprecipitation aids in the presence,

up/down regulation, size, stability, and
interactions of the protein of interest

• IP isolated proteins can then further be

analyzed by Western blotting, ELISA, and
mass spectrometry

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 BASIC OUTLINE OF IMMUNOPRECIPITATION

• It is a technique of precipitating protein antigens out of a

solution using a specific antibody which is immobilized to a
solid support
• IP is based on a solid bead)that contains a binding protein
• IP requires a specific antibody to purify a single antigen
• The sample containing the protein of interest is incubated with
the beads and antibody.
• The antibody binds to the protein and bead
• The beads get washed
• The protein is eluted

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IMMUNOPRECIPITATION - ILLUSTRATION

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 METHODS USED IN IMMUNOPRECIPITATION
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
 TYPES OF IMMUNOPRECIPITATION
 Co-immunoprecipitation

• Used to analyze protein–protein interactions

• Main purpose of Co-IP is the identification of interaction

partners to the protein of interest

• It is a technique of precipitating protein antigens out of a

solution using a specific antibody which is immobilized to a
solid support

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 CO-IMMUNOPRECIPITATION

Co-immunoprecipitation is a well known and popular method

for studying protein-protein ineraction in-vitro.

The main applications are (i) Prove interaction between two

proteins and (ii) IIdentifies a new protein by using a known
one.
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 CHROMATIN IMMUNOPRECIPITATION
• 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
quantization

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CHROMATIN IMMUNOPRECIPITATION
 RNA IMMUNOPRECIPITATION
• 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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COMPARISION OF BEADS IN IMMUNOPRECIPITATION
 FACTORS INFLUENCING IP

• Performing an IP experiment is relatively simple

• Variable factors depend on the protein-of-interest and the
antibody
• The core of an IP experiment is the purification of a specific
protein with specific binding to an antibody

Main Factors Possible Solution

Wash, elution, binding buffer Composition, volume

Type of solid support Physical characteristics

Antibody Amount, specificity
Pre-clearing of lysate Non-specific binding

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 IMMUNOPRECIPITATION - STEPS

Sample Preparation

Use of an Isolate control

Pre-Cleaning of sample

Antibody Incubation

Precipitation of Protein / Protein Complex

Washing and Elution

Analysis of the Precipitate

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 IMMUNOPRECIPITATION PROTOCOL
(I) Sample Preparation
• Prepare lysate from cells according to your routine
protocol
• Commonly used amounts: 0.3–0.5 ml lysate containing
1–4 mg total protein
• Use protease inhibitors when working with RIPA lysate
buffer
• Use sufficient lysates for the first trial. Typically 1–3 mg
total protein is needed for each IP
• Concentration of proteinase inhibitor should be 1.5–2
times of that for western blotting lysates 15
 IMMUNOPRECIPITATION PROTOCOL
(II) Lysate Pre-cleaning
• Resuspend Protein A or G sepharose beads slurry by
gentle vortex, then add 50 μl of 50% beads slurry per
0.5–1 mg of cell lysate
• Incubate at 4°C for 30 min on a rotator
• Centrifuge at 1000 rpm for 3 min at 4°C and transfer the
supernatant to a fresh tube
• Note: Tissues with abundant IgG are suggested to be
pre-cleared with Protein A or G sepharose

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 IMMUNOPRECIPITATION PROTOCOL
(III) IMMUNOPRECIPITATION
• Add appropriate amount of primary antibody to the
whole lysate
• Optimal antibody concentration should be determined
by titration
• Gently rock the mixture at 4°C for 2–4 h or overnight
• Set up a negative control with IgG corresponding to the
primary antibody source
• Add Protein A or G sepharose beads slurry to capture
the immunocomplex
• Gently rock the mixture at 4°C for 1–4 h 17
(III) IMMUNOPRECIPITATION - CONTINUATION
• Centrifuge the mixture at 500–1000 rpm for 30 s at 4°C
and discard the supernatant
• Wash the beads 3–4 times with 1 ml RIPA lysis buffer or
1X PBS with 0.2% Tween 20 (less stringent), centrifuge
and discard the supernatant
(IV) ELUTION
• Elute the pellet twice with 40 μl 0.10 M Glycine, 0.05M
Tris-HCl (pH 1.5–2.5) elution buffer containing 500 mM
NaCl
• Add 5X SDS sample buffer to the elutions. Heat at 95°C
for 5 min 18
 BINDING PROTEINS IN IP
• Protein A, Protein G or Protein A/G are used as
immunoglobulins binding proteins in IP
• Protein A/G binds to all subclasses of immunoglobulins
• Protein A or Protein G bind to multiple subclasses of
immunoglobulins

Species Subclass Protein A Protein G Protein

A/G Binding Capacity
Human IgG1 +++ +++ +++ +++ High
Human IgG2 +++ +++ +++ ++ Good
Human IgM + – + + Low
Mouse IgG1 + ++ ++ – None
Mouse IgG2a +++ +++ +++
Mouse IgG2b +++ +++ +++
Rat IgG1 + ++ ++
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 BUFFERS USED IN IP
(I) 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
• 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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 BUFFERS USED IN IP
(II) Wash Buffers
• Multiple washes with simple wash buffers, such as PBS
either alone or with low detergent concentrations can
be used to remove contaminants
(III) 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 Ab-Protein A/G interaction

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 TROUBLESHOOTING
Issue Possible Solution
Wrong lysis buffer. Change lysis buffer.
No antibody binding to Make sure, isotype specific beads were used.
beads.
Protein of interest cannot Change elution buffer (components, pH etc).
be eluted from the beads.
Insufficient antibody Too low antibody amount, titrate antibody concentration.
amount for binding
properly.
Protein-of-interest is Pre-clear the sample to decrease non-specific binding.
low expressed.

Issue Possible Solution

Insufficient washing. Increase washing volume/time.
Non-specific antibody. Pre-test the antibody for its specifity.
Antibody amount. A too high amount of antibody leads to non-specific binding.
Too high amount of Decrease the amount of cells/lysate.
sample.
Proteins bind non- Reduce sample amount, pre-clear samples.
specific to the
antibody.
Antigen degradations. Add fresh protease inhibitors.

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 APPLICATIONS
• Isolate / Detect Proteins of interest
• Enrichment of low abundant proteins
• Study protein-protein interaction and protein complexes
• Identify unknown proteins in a protein complex
• Verify protein expression in a specific tissue.

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