IMMUNOTECHNIQUES (immunoprecipitation)
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Transcript of IMMUNOTECHNIQUES (immunoprecipitation)
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…..