IMMUNOPRECIPITATION

INTRODUCTION

“Immunoprecipitation is a technique used

to enrich or purify a specific protein from

a complex mixture using an antibody”

• Also provides a sensitive assay for the

presence of a particular antigen, in a cell

or tissue

• It can be used for a wide variety of

applications such as protein function and

studying protein- protein complexes

TYPES OF

IMMUNOPRECIPITATION

INDIVIDUAL PROTEIN

IMMUNOPRECIPITATION

(IP)

PULL DOWN ASSAY

PROTEIN COMPLX

IMMUNOPRECIPITATION

(CO-IP)

INDIVIDUAL PROTEIN

IMMUNOPRECIPITATION

It is an assay designed to purify a single

antigen from a complex mixture using a

specific antibody attached to a beaded

support (Immobilized protein complex).

• A common sequential

method to incubate

antibody and sample (cell

lysate), followed by

addition of affinity beads

(directly or indirectly) A

common sequential

Antibody may be incubate first with

beads

(it become binds with IgG binding

proteins, protein A, G or A/G)

Antigen containing

sample added

Antigen, antibody and

support are binding out

Beads are washed

extensively

Antigen eluted from

support using appropriate

elution buffer

PROTEIN COMPLEX

IMMUNOPRECIPITTATION

It is very similar to IP because it uses an

immobilization antibody specific to an antigen of

interest.

A Co-IP is designed to isolate the antigen along

with any protein or ligands that are bound to it.

Such instances the known antigen is termed as Bait

protein and the proteins interacts with , are called

Prey proteins.

Number of factors which affect the Co-IP-

• Optimization of binding and wash

conditions must include consideration of

effect on Bait- Prey interaction

PULL DOWN ASSAY

It is similar in concept to a Co-IP, performed in order to

investigate protein or ligands that bound to a bait protein.

It proves a suspected interaction between two proteins and

investigate unknown proteins

it is not based on Ab- Ag interaction.

The bait protein capture to solid support (beads) by a non-

antibody affinity system ( by covalent attachment and affinity

tag)

Example- immobilized metal affinity

chromatography (IMAC) can be used

to perform pull down assay with

histidine-tagged bait proteins

METHODS OF

IMMUNOPRECIPITATION

INDIRECT

OR

TRADITIONAL

METHOD

CROSS LINK

METHOD

DIRECT

METHOD

INDIRECT METHOD

Protein A, G

or A/G binding

proteins

Beaded

supportForms Ab- binding

platform

Antibodies

binds with

protein

Protein A, G

or A/G binding

proteins

Beaded

supportAntibody

Beaded support

removed out and

desired protein

immunoprecipitated

DIRECT METHOD

CROSS LINK METHOD

PROCEDURE OF

IMMUNO-

PRECIPITATION

2. Preparation

of immune

complex

4. Elution

of immune

complex

3. Capture of

immune

complex

1. Material

preparation

MATERIAL PREAPRATION

1. IP Lysis / Wash Buffer0.025M Tris, 0.15 M NaCl

0.001 M EDTA

1% NP-40

5% Glycerol

2. Saline solution- 0.15 M NaCl

3. SDS PAGE Sample Buffer

Lane marker reducing sample buffer

dilution

Use 100mM Tris pH 6.8 40mM DTT,

2%SDS, 20% Glycerol, 0.2% brmophenol

blue

4. ELUTION BUFFER

IgG Elution Buffer or 0.1-0.2 M

Glycine. HCl at pH 2.5-3.0

PREPARATION OF IMMUNE

COMPLEX

Prepare protein sample, use the IP Lysis / wash buffer

Use 500ul of IP Lysis / wash buffer per 50mg of wet cell pellet

Combine 2-10ug of affinity purified antibody

with the cell lysate

Total protein/IP REACTION 500-1000ug

Dilute the antibody/ lysate

solution to 300-600ul with IP

Lysis/ wash buffer

Incubate for 1 hours to overnight

at 4°C

Immune complex

formed

CAPTURE OF IMMUNE COMPLEX

Gently shake the water of protein A/G

Agarose to obtain an even suspension

Add 10 ul of resin into spin column

Place the column into micro centrifuge (1000 x

g) for 1 min

Wash the resin twice with 100 ul of cold IP

Lysis/wash buffer.

Gently tap the bottom of spin column to

remove excess liquid and insert the bottom

plug into the spin column

Add the antibody/lysate sample to protein A/G

plus agarose in spin column

Remove the bottom plug

Centrifuge column and save the flow

through

ELUTION OF THE IMMUNE COMPLEX

Sample Buffer Elution• Ideal for Western Blot

analysis

Place the spin column containing

the resin into new collection tube

and add 50 ul 2 x SDS PAGE

• Incubate at 100 C for 5-10

mins

• Centrifuge to collect elute,

cool at room temperature

before apply an SDS PAGE

Gel

Low Buffer Elution• Ideal for enzymatic and

functional assay

• Place the spin column

containing the resin into the

new collection tube and add

50 ul 2 X SDS PAGE

• Incubate for 10 min at room

temperature

• Centrifuge the tube and

collect the flow through

APPLICATIONS

• It is used to estimate the molecular weight,

identity or quantity of a protein of interest.

• Study protein- protein interaction

• Determine the concentration of protein

• Analyse the expression level of protein of

interest

• Studying Cancer development, cell signalling

pathway and diseases

• Post translational modification

• Verify protein expression in a specific tissue.

THANK YOU…..