Feb 16, 2022
Version 2
Immunoprecipitation using Protein A/G Magnetic Beads V.2
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
External link: https://www.neb.com/protocols/0001/01/01/immunoprecipitation-using-protein-ag-magnetic-beads
Protocol Citation: New England Biolabs 2022. Immunoprecipitation using Protein A/G Magnetic Beads. protocols.io https://dx.doi.org/10.17504/protocols.io.bddai22eVersion created by New England Biolabs
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: March 08, 2020
Last Modified: February 16, 2022
Protocol Integer ID: 33922
Keywords: Protein A magnetic beads, Protein G magnetic beads, immunoprecipitating, non-specific binding to beads, pre-clearing crude cell extract of proteins, Immunoprecipitation, IP
Abstract
This protocol explains immunoprecipitation using Protein A/G Magnetic Beads.
Materials
MATERIALS
Sodium Orthovanadate (Vanadate) - 1 mlNew England BiolabsCatalog #P0758S
Protein A Magnetic Beads - 1 mlNew England BiolabsCatalog #S1425S
Protein G Magnetic Beads - 1 mlNew England BiolabsCatalog #S1430S
PMSFMerck MilliporeSigma (Sigma-Aldrich)Catalog #P7626
Bromophenol blueBio Basic Inc.Catalog #BB2230.SIZE.25g
GlycerolBio Basic Inc.Catalog #GB0232.SIZE.500ml
SDSBio Basic Inc.Catalog #SB0485.SIZE.100g
DTT (Dithiothreitol) (> 99% pure) Protease freeGold BiotechnologyCatalog #DTT
EGTAGold BiotechnologyCatalog #E-217
Triton X-100 Merck MilliporeSigma (Sigma-Aldrich)Catalog #93426
Tris-HClLife TechnologiesCatalog #AM9855
2-MercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3148
EDTAFisher ScientificCatalog #16 004Y
Immunoprecipitation Buffer:
| A | B | |
| NaCl | 150 mM | |
| Tris-HCl (pH 7.4) | 10 mM | |
| EDTA | 1 mM | |
| EGTA (pH 8.0) | 1 mM | |
| Sodium ortho-vanadate | 0.2 mM | |
| PMSF | 0.2 mM | |
| Triton X-100 | 1% | |
| NP-40 | 0.50% |
3X SDS Sample Loading Buffer:
| A | B | |
| Tris-HCl (pH 6.8) | 187.5 mM | |
| SDS | 6%(w/v) | |
| Glycerol | 30% | |
| DTT | 150 mM | |
| Bromophenol blue | 0.03% (w/v) | |
| β-mercaptoethanol) | 2% |
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Before start
Use 25 µl of Protein A/G Magnetic Beads per 200 µl of crude cell lysate containing 200-500 µg of total protein in a standard immunoprecipitation protocol. It is important to increase the volume of beads proportionately for larger cell lysate volumes.
Prepare Immunoprecipitation buffer with the following reagents:
| A | B | |
| NaCl | 150 mM | |
| Tris-HCl (pH 7.4) | 10 mM | |
| EDTA | 1 mM | |
| EGTA (pH 8.0) | 1 mM | |
| Sodium ortho-vanadate | 0.2 mM | |
| PMSF | 0.2 mM | |
| Triton X-100 | 1% | |
| NP-40 | 0.50% |
Prepare 3X SDS Sample Loading Buffer using the following reagents:
| A | B | |
| Tris-HCl (pH 6.8) | 187.5 mM | |
| SDS | 6%(w/v) | |
| Glycerol | 30% | |
| DTT | 150 mM | |
| Bromophenol blue | 0.03% (w/v) | |
| β-mercaptoethanol) | 2% |
Cell Lysis
Cell Lysis
Rinse a 60 mm culture dish of confluent cells with PBS.
Lyse the cells with 0.5 mL cold Immunoprecipitation Buffer .
Note
Immunoprecipitation buffer is prepared with the following reagents:
| A | B | |
| NaCl | 150 mM | |
| Tris-HCl (pH 7.4) | 10 mM | |
| EDTA | 1 mM | |
| EGTA (pH 8.0) | 1 mM | |
| Sodium ortho-vanadate | 0.2 mM | |
| PMSF | 0.2 mM | |
| Triton X-100 | 1% | |
| NP-40 | 0.50% |
Maintain constant agitation for 00:30:00 at 4 °C .
Scrape the cells from the dish.
Sonicate On ice for 00:00:05 ; repeat 4 more times:
Sonicate On ice for 00:00:05 (1/4).
Sonicate On ice for 00:00:05 (2/4).
Sonicate On ice for 00:00:05 (3/4).
Sonicate On ice for 00:00:05 (4/4).
Centrifuge for 00:05:00 at 4 °C .
Assay for total protein then adjust concentration to approximately 1 mg/ml with Immunoprecipitation Buffer.
Note
The supernatant is the crude cell lysate.
Immunoprecipitation
Immunoprecipitation
In a 1.5 ml microcentrifuge tube, add 25 µL protein A/G Magnetic Beads to 200 µL crude cell extract .
Note
Steps 8-12 pre-clear crude cell extract of proteins which can bind non-specifically to the beads.
Gently vortex.
Incubate at 4 °C for 01:00:00 .
Apply magnetic field for 00:00:30 to pull beads to the side of the tube.
Pipette supernatant to a clean 1.5 ml microcentrifuge tube and discard the beads.
Add 1 µg -5 µg of desired antibody to crude cell lysate.
Vortex.
Incubate at 4 °C for 01:00:00 .
Note
If monoclonal antibodies are used, add 5 µg rabbit anti-mouse IgG antibody . Vortex and incubate an additional 00:30:00 at 4 °C . Alternatively, Protein G Magnetic Beads (NEB #S1430S) can be used for immunoprecipitations with monoclonal antibodies.
Add 25 µL Protein A/G Magnetic Beads suspension .
Gently vortex.
Incubate with agitation for 01:00:00 at 4 °C .
Apply magnetic field to pull beads to the side of the tube.
Carefully pipette to remove supernatant.
Wash with 500 µL Immunoprecipitation Buffer by gentle vortex.
Apply magnetic field, then remove supernatant and discard.
Repeat wash steps two more times:
Wash with 500 µL Immunoprecipitation Buffer by gentle vortex. (1/2)
Apply magnetic field, then remove supernatant and discard. (1/2)
Wash with 500 µL Immunoprecipitation Buffer by gentle vortex. (2/2)
Apply magnetic field, then remove supernatant and discard. (2/2)
Resuspend bead pellet in 30 µL 3X SDS Sample Loading Buffer .
Note
3X SDS Sample Loading Buffer is prepared using the following reagents:
| A | B | |
| Tris-HCl (pH 6.8) | 187.5 mM | |
| SDS | 6%(w/v) | |
| Glycerol | 30% | |
| DTT | 150 mM | |
| Bromophenol blue | 0.03% (w/v) | |
| β-mercaptoethanol) | 2% |
Incubate sample at 70 °C for 00:05:00 .
Apply magnetic field to sample, then load supernatant on SDS-PAGE gel and electrophorese.