PureProteome Crosslink Antibodies - Millipore

Protocol guide
Immunoprecipitation by
crosslinking antibodies to
PureProteome Protein A, G
or A/G mix magnetic beads
Crosslinking immunoprecipitation:
antigen elution without antibody
interference
Protein A and G magnetic beads are routinely used in
immunoprecipitation (IP) experiments. In traditional IP,
the capture antibody binds reversibly to immobilized
protein A or G and can be easily eluted along with
the target using pH changes or denaturing elution
conditions. In some instances, this coelution of the
capture antibody can interfere with immunodetection,
when the antibody’s heavy or light chain obscures
the target protein. Using crosslinking IP (Figure 1)
can overcome this challenge by eliminating antibody
coelution.
The Traditional IP Method
Magnetic
Bead
Magnetic
Bead
Magnetic
Bead
Magnetic
Bead
Discard
Protein
A/G
Protein
A/G
Protein
A/G
Protein
A/G
Antigen
Sample
Boil in
SDS-PAGE
Sample
Buffer
Antibody
The Crosslink Method
Magnetic
Bead
Magnetic
Bead
Magnetic
Bead
Magnetic
Bead
Reuse
is possible
Protein
A/G
Protein
A/G
Protein
A/G
Protein
A/G
Elution
Buffer
Antibody
Crosslinker
Antigen
Sample
Benefits of PureProteome
Magnetic Beads
• Highest binding capacity:
- Protein A binds 1.5-2.5 mg of rabbit IgG per mL
of suspension
- Protein G binds 2.5-3.5 mg of rabbit IgG per mL
of suspension
- Protein A/G mix binds 2-3 mg of rabbit
IgG per mL of suspension
• Low nonspecific binding
• Economical: Up to half the price of competitor
magnetic beads
HC
LC
Antibody
Reference
Traditional
IP Products
Electrophoresis
and staining
Crosslink IP
Product
Figure 1.
Crosslinking IP (right hand side) can improve
detection of target protein without interference from
antibody chain bands, which are frequently seen in
traditional IP (left hand side).
Target Protein
EMD Millipore is a division of Merck KGaA, Darmstadt, Germany

To avoid coelution, the capture antibody may be
crosslinked to the protein A or G immobilized on the
PureProteome magnetic beads. Commonly used
crosslinking agents include BS 3 (Bis(sulfosuccinimidyl)
suberate), DSS (disuccinimidyl suberate) and DMP
(dimethyl pimelimidate). These homobifunctional aminereactive
crosslinkers react with the primary amines
of the immobilized protein A or G and the capture
antibody, forming a stable amide bond. While BS 3 and
DMP are water-soluble, DSS has to be initially dissolved
in dimethyl sulfoxide (DMSO) or dimethyl formamide
(DMF); once dissolved it can be diluted to the working
concentration using a non-amine-containing buffer.
Here, we describe strategies for covalent crosslinking of
antibody to the PureProteome protein A, G or A/G mix
magnetic mix beads. The methods provided are guidelines
and may be further optimized for individual applications.
Materials
PureProteome Protein A Magnetic Beads,
Catalogue No. LSKMAGA10 or LSKMAGA02
PureProteome Protein G Magnetic Beads,
Catalogue No. LSKMAGG10 or LSKMAGG02
PureProteome Protein A/G Mix Magnetic Beads,
Catalogue No. LSKMAGAG10 or LSKMAGAG02
PureProteome Magnetic Stand,
Catalogue No. LSKMAGS08
Antibody of choice (typically 4-10 µg per reaction)
Coupling buffer for crosslinking with BS 3 and DSS:
20 mM sodium phosphate
0.15 M NaCl (pH should be adjusted to 7-9)
Coupling buffer for crosslinking with DMP:
0.2 M triethanolamine pH 8.2-9
Wash/storage buffer (PBS-T):
Phosphate-buffered saline (PBS) pH 7.4
0.01-0.05% Tween® surfactant 20
Quench buffer:
1M Tris HCl pH 7.5
Crosslinker:
BS 3 bis(sulfosuccinimidyl) suberate, ProteoChem
Catalogue No. c1103-100mg or equivalent
DSS (disuccinimidyl suberate,) ProteoChem
Catalogue No. c1105-1gm or equivalent
DMP (Dimethyl pimelimidate),
Sigma Catalogue No. D-8388 or equivalent
Sample Results
Crosslinking of antibody to PureProteome
magnetic beads improve IP results without
antibody contamination
Figure 2 shows that compared to results of standard IP
(“Std. IP”), immunoprecipitation using PureProteome
protein G magnetic beads crosslinked to anti-GAPDH
results in a single, clear signal corresponding to the
protein of interest. Standard IP lanes also show signals
corresponding to the antibody heavy and light chains. The
bottom panel of Figure 2 shows that, although the ERK1/2
protein is abundant in the lysate input, no ERK1/2 signal
is seen in immunoprecipitated complexes, indicating that
PureProteome protein G magnetic beads demonstrate
low nonspecific binding.
A
B
Lysate BS 3 DSS DMP
Std. IP
Std. IP
Std. IP
Bead +
Antibody
No IP
IgG
Heavy
Chain
GAPDH
IgG
Light
Chain
ERK1/2
Figure 2.
Immunoprecipitation of GAPDH from Jurkat cell lysate
using PureProteome protein G magnetic beads crosslinked
to the anti-GAPDH capture antibody using either BS 3 ,
DSS or DMP crosslinking agents (A). Immunoprecipitated
complexes from (A) were probed with anti-ERK1/2 to assess
nonspecific binding of proteins to PureProteome protein G
magnetic beads (B).
2

Protocol 1
Antibody binding to
PureProteome protein A, G or
A/G mix magnetic beads
(for more detailed instructions refer to user guide)
1. Resuspend magnetic beads by vortexing, ensuring that all
of the beads are uniformly resuspended.
2. Pipette 25-50 µL of bead slurry into a 1.5 mL
microcentrifuge tube. Place tube into magnetic stand to
capture the beads. Remove storage buffer with pipette
and discard.
3. Disengage the magnet from the stand and add 500 µL
wash buffer to the beads. Vortex for 10 seconds. Reengage
magnet to capture the beads and discard wash
buffer. Repeat two more times.
4. Disengage the magnet and resuspend the washed beads
in 100 μL PBS-T. Add capture antibody (typically 4-10 μg)
to the resuspended beads.
5. Allow antibody to bind to beads for 10-30 minutes at
room temperature with continuous mixing. If performing
this step at 4 °C, allow antibody to bind for 60 minutes.
6. Re-engage the magnet to capture the beads. Remove the
buffer containing unbound antibody; if necessary, retain
for later analysis.
7. Disengage magnet. Wash beads by adding 500 µL PBS-T,
vortex beads for 10 seconds. Repeat 2 more times for a
total of 3 washes.
8. Engage the magnet to capture beads, remove PBS-T, then
add 500 µL coupling buffer. Remove the magnet and
vortex beads for 60 seconds. Insert magnet, collect beads
and discard buffer. Repeat two more times.
9. Beads are ready for crosslinking.
Protocol 2
Antibody crosslinking using BS 3
Note: Crosslinker solution should be prepared immediately
before use and should not be stored for later use.
1. Each crosslinking reaction requires 250 µL of 5 mM
crosslinker solution.
2. Prepare a 100 mM BS 3 stock solution by weighing out
2 mg of BS 3 and dissolving in 35 µL Milli-Q® water.
3. Once dissolved, dilute to 5 mM by adding 665 µL
coupling buffer.
4. Add 250 µL of 5 mM BS 3 solution to each crosslinking
reaction and incubate with end-over-end mixing for 30-
60 minutes at room temperature.
5. To quench the crosslinker, add 12.5 µL of quench buffer
to each reaction, incubate for 30-60 minutes at room
temperature with end-over-end mixing.
6. Engage magnet to capture the beads, then remove the
solution with a pipette and discard. Disengage magnet.
7. Wash beads 1x 500 µL for 1 minute using 0.2 M glycine
HCl, pH 2.5 to remove any non-crosslinked antibody.
Engage magnet to capture beads, then remove glycine
solution with pipette and discard.
8. Wash beads for 1 minute using 500 µL PBS-T.
Repeat 2 more times for a total of 3 washes.
9. Use beads immediately in IP experiments or store at
4 °C. For long-term storage, it is recommended that a
bacteriostat such as sodium azide (0.05%) is added to the
bead storage buffer to prevent microbial growth.
Protocol 3
Antibody crosslinking using DSS
Note: Crosslinker solution should be prepared immediately
before use and should not be stored for later use.
1. Each crosslinking reaction requires 250 µL of 5 mM
crosslinker solution.
2. Prepare a 100 mM DSS stock solution by weighing out
2 mg of DSS and dissolving in 54 µL DMSO or DMF.
3. Once dissolved, dilute to 5 mM by adding 646 µL
coupling buffer.
4. Add 250 µL of 5 mM DSS solution to each crosslinking
reaction and incubate with end-over-end mixing for
30-60 minutes at room temperature.
5. To quench the crosslinker, add 12.5 µL of quench buffer to
each reaction and incubate for 30-60 minutes at room
temperature with end-over-end mixing.
6. Place tube into magnetic stand to capture beads, remove
solution with a pipette and discard.
7. Wash beads for 1 minute with 500 μL 0.2 M glycine HCl,
pH 2.5 to remove any non-crosslinked antibody. Engage
magnet to capture beads and discard solution.
8. Wash beads for 1 minute using 500 µL PBS-T. Repeat 2 more
times for a total of 3 washes.
9. Use beads immediately in IP experiments or store at 4 °C.
For long-term storage, it is recommended that a bacteriostat
such as sodium azide (0.05%) is added to the bead storage
buffer to prevent microbial growth.
Protocol 4
Antibody crosslinking using DMP
Note: Crosslinker solution should be prepared immediately
before use and should not be stored for later use.
1. Each crosslinking reaction requires 500 µL of 20 mM
crosslinker solution.
2. Prepare 20 mM DMP solution by weighing out 4 mg DMP
and dissolving in 772 µL of 0.2 M triethanolamine coupling
buffer.
3. Add 500 µL of 20 mM DMP solution to each crosslinking
reaction and incubate with end-over-end mixing for
60 minutes at room temperature.
4. To quench the crosslinker, add 50 µL of quench buffer to
each reaction and incubate for 30-60 minutes at room
temperature with end-over-end mixing.
5. Place tube into magnetic stand, collect beads and remove
solution.
6. Wash beads for 1 minute with 500 μL 0.2 M glycine HCl, pH
2.5 to remove any non-crosslinked antibody. Engage magnet
to capture beads and discard solution.
7. Wash beads for 1 minute using 500 μL PBS-T.
Repeat 2 more times for a total of 3 washes.
8. Use beads immediately in IP experiments or store at
4 °C. For long-term storage, it is recommended that a
bacteriostat such as sodium azide (0.05%) is added to the
bead storage buffer to prevent microbial growth.
3

Figure 3.
The PureProteome
Magnetic Stand is
designed to rapidly and
easily isolate magnetic
particles from up to eight
1.5 mL or 2.0 mL tubes.
The stand features a
removable magnet and
unique vortex interface
that enables thorough
mixing without having to
remove tubes from the
stand.
Ordering Information
Description Qty/Pk Catalogue No.
PureProteome Protein A Magnetic Beads 2 x 1 mL LSKMAGA02
1 x 10 mL LSKMAGA10
PureProteome Protein G Magnetic Beads 2 x 1 mL LSKMAGG02
1 x 10 mL LSKMAGG10
PureProteome Protein A/G Mix Magnetic Beads 2 x 1 mL LSKMAGAG02
1 x 10 mL LSKMAGAG10
PureProteome Magnetic Stand, 8-well 1 LSKMAGS08
To Place an Order or Receive
Technical Assistance
In the U.S. and Canada, call toll-free 1-800-645-5476
For other countries across Europe and the world,
please visit: www.emdmillipore.com/offices
For Technical Service, please visit:
www.emdmillipore.com/techservice
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EMD Millipore, the M logo, and PureProteome are trademarks
and Milli-Q is a registered trademark of Merck KGaA, Darmstadt, Germany.
Trademarks belonging to third parties are the properties of their respective owners.
Lit No. PC5522EN00 BS-GEN-13-07871 03/2013 Printed in the USA.
© 2013 EMD Millipore Corporation, Billerica, MA USA. All rights reserved.