IMMUNOTECHNIQUES (immunoprecipitation) for M.Sc. students

1. IMMUNOPRECIPITATION

2. 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

3. TYPES OF
IMMUNOPRECIPITATION
INDIVIDUAL PROTEIN
IMMUNOPRECIPITATION
(IP)
PULL DOWN ASSAY
PROTEIN COMPLX
IMMUNOPRECIPITATION
(CO-IP)

4. 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

5. 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

6. 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

7. 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

8. METHODS OF
IMMUNOPRECIPITATION
INDIRECT
OR
TRADITIONAL
METHOD
CROSS LINK
METHOD
DIRECT
METHOD

9. INDIRECT METHOD
Protein A, G
or A/G binding
proteins
Beaded
support
Forms Ab- binding
platform
Antibodies
binds with
protein
Protein A, G
or A/G binding
proteins
Beaded
support
Antibody
Beaded support
removed out and
desired protein
immunoprecipitated

10. DIRECT METHOD

11. CROSS LINK METHOD

12. PROCEDURE OF
IMMUNO-
PRECIPITATION
2. Preparation
of immune
complex
4. Elution
of immune
complex
3. Capture of
immune
complex
1. Material
preparation

13. MATERIAL PREAPRATION
1. IP Lysis / Wash Buffer
0.025M 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

14. 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

15. 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

16. 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

17. 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.

18. THANK YOU…..