TITLE: Immunoprecipitation using Protein A/G Magnetic Beads
AUTHORS: New  England   Biolabs

[DESCRIPTION]
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: 
      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: 
      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]
SECTION: Cell Lysis
1. Rinse a 60 mm culture dish of confluent cells with PBS.
SECTION: Cell Lysis
2. Lyse the cells with 0.5 mL. 
 

SECTION: Cell Lysis
3. Maintain constant agitation for 30 min at 4 °C.
SECTION: Cell Lysis
4. Scrape the cells from the dish.
SECTION: Cell Lysis
5. Sonicate on ice for 5 s; repeat 4 more times:

SECTION: Cell Lysis
6. Centrifuge for 5 min at 4 °C.
SECTION: Cell Lysis
7. Assay for total protein then adjust concentration to approximately 1 mg/ml with Immunoprecipitation Buffer.
 

SECTION: Immunoprecipitation
8. In a 1.5 ml microcentrifuge tube, add 25 µL to 200 µL.
 

SECTION: Immunoprecipitation
9. Gently vortex.
SECTION: Immunoprecipitation
10. Incubate at 4 °C for 60 min. 
SECTION: Immunoprecipitation
11. Apply magnetic field for 30 s to pull beads to the side of the tube.
SECTION: Immunoprecipitation
12. Pipette supernatant to a clean 1.5 ml microcentrifuge tube and discard the beads.
SECTION: Immunoprecipitation
13. Add 1 µg-5 µg of desired antibody to crude cell lysate.
SECTION: Immunoprecipitation
14. Vortex. 
SECTION: Immunoprecipitation
15. Incubate at 4 °C for 60 min.
 
SECTION: Immunoprecipitation
16. Add 25 µL.
SECTION: Immunoprecipitation
17. Gently vortex.
SECTION: Immunoprecipitation
18. Incubate with agitation for 60 min at 4 °C.
SECTION: Immunoprecipitation
19. Apply magnetic field to pull beads to the side of the tube.
SECTION: Immunoprecipitation
20. Carefully pipette to remove supernatant.
SECTION: Immunoprecipitation
21. Wash with 500 µL by gentle vortex.
SECTION: Immunoprecipitation
22. Apply magnetic field, then remove supernatant and discard. 
SECTION: Immunoprecipitation
23. Repeat wash steps two more times: 
SECTION: Immunoprecipitation
24. Resuspend bead pellet in 30 µL.
 

SECTION: Immunoprecipitation
25. Incubate sample at 70 °C for 5 min.
SECTION: Immunoprecipitation
26. Apply magnetic field to sample, then load supernatant on SDS-PAGE gel and electrophorese.
SECTION: Cell Lysis
5.1. Sonicate on ice for 5 s (1/4).
SECTION: Cell Lysis
5.2. Sonicate on ice for 5 s (2/4).
SECTION: Cell Lysis
5.3. Sonicate on ice for 5 s (3/4).
SECTION: Cell Lysis
5.4. Sonicate on ice for 5 s (4/4).
SECTION: Immunoprecipitation
23.1. Wash with 500 µL by gentle vortex. (1/2)
SECTION: Immunoprecipitation
23.2. Apply magnetic field, then remove supernatant and discard. (1/2)
SECTION: Immunoprecipitation
23.3. Wash with 500 µL by gentle vortex. (2/2)
SECTION: Immunoprecipitation
23.4. Apply magnetic field, then remove supernatant and discard. (2/2)