Immunoprecipitation using Protein A/G Magnetic Beads
New England Biolabs
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.
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% |
Steps
Cell Lysis
Rinse a 60 mm culture dish of confluent cells with PBS.
Lyse the cells with 0.5mL.
Maintain constant agitation for 0h 30m 0s at 4°C.
Scrape the cells from the dish.
Sonicate On ice for 0h 0m 5s; repeat 4 more times:
Sonicate On ice for 0h 0m 5s (1/4).
Sonicate On ice for 0h 0m 5s (2/4).
Sonicate On ice for 0h 0m 5s (3/4).
Sonicate On ice for 0h 0m 5s (4/4).
Centrifuge for 0h 5m 0s at 4°C.
Assay for total protein then adjust concentration to approximately 1mg/ml with Immunoprecipitation Buffer.
Immunoprecipitation
In a 1.5 ml microcentrifuge tube, add 25µL to 200µL.
Gently vortex.
Incubate at 4°C for 1h 0m 0s.
Apply magnetic field for 0h 0m 30s to pull beads to the side of the tube.
Pipette supernatant to a clean 1.5 ml microcentrifuge tube and discard the beads.
Add 1µg-5µg of desired antibody to crude cell lysate.
Vortex.
Incubate at 4°C for 1h 0m 0s.
Add . 25µL.
Gently vortex.
Incubate with agitation for 1h 0m 0s at 4°C.
Apply magnetic field to pull beads to the side of the tube.
Carefully pipette to remove supernatant.
Wash with 500µL by gentle vortex.
Apply magnetic field, then remove supernatant and discard.
Repeat wash steps two more times:
Wash with 500µL by gentle vortex. (1/2)
Apply magnetic field, then remove supernatant and discard. (1/2)
Wash with 500µL by gentle vortex. (2/2)
Apply magnetic field, then remove supernatant and discard. (2/2)
Resuspend bead pellet in 30µL.
Incubate sample at 70°C for 0h 5m 0s.
Apply magnetic field to sample, then load supernatant on SDS-PAGE gel and electrophorese.
