CST Guide:

15
Section II: ANTIBODY APPLICATIONS
For targets that run
near 50 kDa, use
light chain-specific or
conformation-specific
secondary antibody to
avoid obscuring the
intended target band.
Immunoprecipitation (IP)
Protein Enrichment
Immunoprecipitation (IP) uses antibodies bound to protein A or G beads to isolate target proteins from
a cell lysate. IP is often used as an enrichment strategy to study low abundance proteins that cannot
be detected directly by western blot. IP is also used to identify members of protein complexes, as
proteins associated with the target protein will co-precipitate with the antibody-bound beads. Proteins
are either eluted off the beads prior to western blot, or the bead-bound protein can be directly analyzed
by western blot.
The stringency of IP buffers is key for experimental success. High stringency buffers that contain
anionic detergents such as SDS may work for IP but are not recommended for co-IP experiments, as
they dissociate protein complexes. Medium and low stringency buffers that contain nonionic detergents
such as NP-40 or Triton X-100 are better suited for co-IP experiments because they allow protein
associations to remain intact. Successful IP and co-IP experiments require specific and sensitive antibodies
to minimize enrichment of non-specific proteins. While unconjugated antibodies are commonly
used for IP experiments, alternative detection options include biotinylated primary antibodies combined
with streptavidin beads or primary antibodies directly conjugated to IP beads. IP beads are available in
agarose, Sepharose ® , or magnetic-bead formats. Agarose and Sepharose beads have a higher protein
binding capacity, but require multiple centrifugation steps, which increases experimental time and
can lead to loss of beads due to pipetting. Magnetic beads are a convenient option as the beads are
precipitated using a magnetic rack, thereby eliminating several centrifugation steps.
A common challenge presented by IP analysis is the presence of an IgG chain band obscuring the
western blot band of interest. The tips below offer options to clarify your results by avoiding crossreacting
IgG bands.
IP Tips for Success
Use conformation-specific secondary antibodies for WB analysis
of an IP if the target size is similar to an IgG subunit size.
The antibody used for IP remains in the sample and often appears on the subsequent western blot as
bands at 50 kDa (IgG heavy chain) and 25 kDa (IgG light chain). The conformation-specific secondary
antibody does not recognize denatured heavy or light IgG chains, so the 25 and 50 kDa bands do not
appear on the western blot. The light chain-specific secondary antibody only recognizes the IgG light
chain, so the 50 kDa heavy chain does not appear on the blot.
kDa
140
100
80
60
50
40
30
20
A B C
1 2 1 2 1 2
Heavy
Chain
PRAS40
Where possible, use primary antibodies
from different species for the IP and the WB.
IgG chains from the IP antibody are recognized by the secondary antibody when the western blot
primary antibody is raised in the same host species (For example, the IgG chains will be detected by
the anti-rabbit secondary antibody when rabbit antibodies are used for IP and western blot). Using IP
and western blot antibodies raised in different species avoids secondary antibody recognition of the IgG
heavy chain and light chain from the IP.
234 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys.
Light
Chain
Mouse Anti-rabbit IgG (Light-Chain Specific) (L57A3) mAb
#3677: IP of PRAS40 from untreated HeLa cells using PRAS40 (D23C7)
Rabbit mAb #2691. Secondary antibodies include Mouse Anti-rabbit
IgG (Conformation Specific) (L27A9) mAb #3678 (A), #3677 (B), and
Anti-rabbit IgG, HRP-linked Antibody #7074 (C). The bound Mouse
Anti-Rabbit IgG mAb was detected by Anti-mouse IgG, HRP-linked
Antibody #7076 (A,B). The positions of the reduced and denatured
rabbit IgG heavy and light chains are indicated.
Lanes
1. 10% input of untreated HeLa cells
2. IP of PRAS40 from untreated HeLa cells using PRAS40 (D23C7)
Rabbit mAb #2691
Akt (pan) (C67E7) Rabbit mAb #4691 and Akt
(pan) (40D4) Mouse mAb #2920: IP of Akt from 293
cells treated with Human Insulin-like Growth Factor I
(hIGF-I) #8917. WB analysis performed with #4691 and
Anti-rabbit IgG, HRP-linked Antibody #7074 (A) or with
#2920 and Anti-mouse IgG, HRP-linked Antibody #7076
(B). Note in lane 5 that the rabbit IgG heavy chain is
recognized by #7074 (A), but not by #7076 (B).
Lanes
1. Beads and 293 + hIGF-I lysate control
2. Rabbit IgG control
3. Mouse IgG control
4. 10% input of 293 + hIGF-I lysate (no IP)
5. IP: Akt (pan) (C67E7) Rabbit mAb #4691
kDa
140
100
80
60
50
40
30
IP Native Protein Protocol
This protocol is intended for IP of native proteins for analysis by western immunoblotting or kinase activity.
20
A
B
1 2 3 4 5 1 2 3 4 5
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L of 1X PBS, add 50 ml 20X PBS to 950 ml dH 2
O, mix.
2. 10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH 2
O, mix.
NOTE: Add 1 mM PMSF (#8553) immediately prior to use.
3. 3X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer
by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer.
4. Protein A or G Agarose Beads (For unconjugated primary antibodies): Use Protein A (#9863, #8687) for rabbit IgG IP
and Protein G (#8740) for mouse IgG IP. NOTE: Magnetic beads (#8687, #8740) require 6-Tube Magnetic Separation Rack
(#7017).
5. Immobilized Streptavidin (Bead Conjugate) (For biotinylated antibodies): (#3419) Gently vortex vial and use 10 µl per IP.
6. 10X Kinase Buffer (for kinase assays): (#9802) To prepare 1 ml of 1X kinase buffer, add 100 µl 10X kinase buffer to
900 µl dH 2 O, mix.
7. ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 µM), add 10 µl ATP (10 mM) to 490 µl 1X
kinase buffer.
B. Preparing Cell Lysates
1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.
3. Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer to each plate (10 cm) and incubate on ice for 5 min.
4. Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice.
5. Sonicate on ice 3 times for 5 sec each.
6. Microcentrifuge for 10 min at 4°C, 14,000 x g and transfer the supernatant to a new tube. The supernatant is the cell lysate.
If necessary, lysate can be stored at -80°C.
C. Immunoprecipitation
Cell Lysate Pre-Clearing (Optional step for unconjugated and biotinylated antibodies.)
1. Add 10–30 µl of 50% bead slurry, either Protein A or G agarose or magnetic beads (for unconjugated primary antibodies) or
10 µl streptavidin beads (#3419; for biotinylated antibodies), to 200 µl cell lysate at 1 mg/ml.
2. Incubate with rotation at 4°C for 30–60 min.
3. Microcentrifuge for 10 min at 4°C. Transfer the supernatant to a fresh tube.
4. Proceed to one of the following specific set of steps depending on the primary antibody used.
Using Unconjugated Primary Antibodies
1. Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate at 1 mg/ml.
Incubate with gentle rocking overnight at 4°C.
2. Add either protein A or G agarose or magnetic beads (10–30 µl of 50% bead slurry). Incubate with gentle rocking for 1–3 hr
at 4°C for agarose beads, or 10–30 min for magnetic beads.
3. Microcentrifuge for 30 sec at 4°C. Wash pellet five times with 500 µl of 1X cell lysis buffer. Keep on ice between washes.
4. Proceed to Analyze by Western Immunoblotting or Analyze by Kinase Assay (Section D).
Using Biotinylated Primary Antibodies
1. Add biotinylated antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate
at 1 mg/ml. Incubate with gentle rocking overnight at 4°C.
2. Gently mix Immobilized Streptavidin (Sepharose ® Bead Conjugate #3419) and add 10 µl of slurry. Incubate with gentle
rocking for 2 hr at 4°C.
3. Microcentrifuge for 30 sec at 4°C. Wash pellet 5 times with 500 µl of 1X cell lysis buffer. Keep on ice during washes.
4. Proceed to Sample Analysis (Section D).
chapter 15: Immunoprecipitation (IP)
Akt
IgG
heavy
chain
IgG
light
chain
Use a mouse derived
western blot antibody
to avoid recognition
of IP antibody rabbit
IgG chains.
Use magnetic beads
to reduce protocol
time by skipping
centrifugation steps.
kDa
140
100
80
60
50
40
30
20
1 2
HC
Syntaxin 6
Protein A Magnetic Beads #8687:
IP of Syntaxin 6 from COS-7 cells using
Syntaxin 6 (C34B2) Rabbit mAb #2869
and #8687. WB analysis was performed
using Syntaxin 6 (C34B2) Rabbit mAb
#2869. Magnetic beads shorten the IP
protocol by eliminating centrifugation
steps after washing and also reduce loss
of beads due to pipetting.
Lanes
1. IP pellet
2. IP supernatant
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