You are on page 1of 4

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 Protein A/G Antibody

The Crosslink Method
Magnetic Bead Protein A/G Antibody

Magnetic Bead

Protein A/G

Magnetic Bead

Protein A/G Crosslinker

Magnetic Bead

Protein A/G

Magnetic Bead

Antigen Sample

Protein A/G

Protein A/G

Magnetic Bead

Boil in SDS-PAGE Sample Buffer

Magnetic Bead

Protein A/G

Antigen Sample

Discard

Reuse is possible

Elution Buffer

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

Electrophoresis and staining HC LC Antibody Reference Traditional IP Products Crosslink IP Product

Target Protein

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).

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 BS3 (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 BS3 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.

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.
Bead + Antibody No IP IgG Heavy Chain GAPDH IgG Light Chain B ERK1/2

Lysate BS3
Std. IP

DSS
Std. IP

DMP
Std. IP

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 BS3 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: BS3 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

A

Figure 2. Immunoprecipitation of GAPDH from Jurkat cell lysate using PureProteome™ protein G magnetic beads crosslinked to the anti-GAPDH capture antibody using either BS3, 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. 2. Resuspend magnetic beads by vortexing, ensuring that all of the beads are uniformly resuspended. 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. 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. Disengage the magnet and resuspend the washed beads in 100 µL PBS-T. Add capture antibody (typically 4-10 µg) to the resuspended beads. 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. Re-engage the magnet to capture the beads. Remove the buffer containing unbound antibody; if necessary, retain for later analysis. 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. 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. Beads are ready for crosslinking.

Protocol 3

Antibody crosslinking using DSS

3.

4.

5.

6.

7.

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.

8.

Protocol 4

Antibody crosslinking using DMP

9.

Protocol 2

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 BS3 stock solution by weighing out 2 mg of BS3 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 BS3 solution to each crosslinking reaction and incubate with end-over-end mixing for 3060 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.

Antibody crosslinking using BS3

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 PureProteome™ Protein G Magnetic Beads PureProteome™ Protein A/G Mix Magnetic Beads PureProteome™ Magnetic Stand, 8-well

2 x 1 mL 1 x 10 mL 2 x 1 mL 1 x 10 mL 2 x 1 mL 1 x 10 mL 1

LSKMAGA02 LSKMAGA10 LSKMAGG02 LSKMAGG10 LSKMAGAG02 LSKMAGAG10 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

Get Connected!
Join EMD Millipore Bioscience on your favorite social media outlet for the latest updates, news, products, innovations, and contests! facebook.com/EMDMilliporeBioscience

www.emdmillipore.com/offices
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.

twitter.com/EMDMilliporeBio