Immunoprecipitation using Protein A/G Magnetic Beads

New England Biolabs

Published: 2022-02-16 DOI: 10.17504/protocols.io.bddai22e

pre-clearing crude cell extract of proteins

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.

Note

Immunoprecipitation buffer is prepared 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%

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:

5.1.

Sonicate On ice for 0h 0m 5s (1/4).

5.2.

Sonicate On ice for 0h 0m 5s (2/4).

5.3.

Sonicate On ice for 0h 0m 5s (3/4).

5.4.

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.

Note

The supernatant is the crude cell lysate.

Immunoprecipitation

In a 1.5 ml microcentrifuge tube, add 25µL to 200µL.

Note

Steps 8-12 pre-clear crude cell extract of proteins which can bind non-specifically to the beads.

Gently vortex.

10.

Incubate at 4°C for 1h 0m 0s.

11.

Apply magnetic field for 0h 0m 30s to pull beads to the side of the tube.

12.

Pipette supernatant to a clean 1.5 ml microcentrifuge tube and discard the beads.

13.

Add 1µg-5µg of desired antibody to crude cell lysate.

14.

Vortex.

15.

Incubate at 4°C for 1h 0m 0s.

Note

If monoclonal antibodies are used, add 5µg. Vortex and incubate an additional 0h 30m 0s at 4°C. Alternatively, Protein G Magnetic Beads (If monoclonal antibodies are used, add . Vortex and incubate an additional at . Alternatively, Protein G Magnetic Beads (NEB #S1430S) can be used for immunoprecipitations with monoclonal antibodies.) can be used for immunoprecipitations with monoclonal antibodies.

16.

Add . 25µL.

17.

Gently vortex.

18.

Incubate with agitation for 1h 0m 0s at 4°C.

19.

Apply magnetic field to pull beads to the side of the tube.

20.

Carefully pipette to remove supernatant.

21.

Wash with 500µL by gentle vortex.

22.

Apply magnetic field, then remove supernatant and discard.

23.

Repeat wash steps two more times:

23.1.

Wash with 500µL by gentle vortex. (1/2)

23.2.

Apply magnetic field, then remove supernatant and discard. (1/2)

23.3.

Wash with 500µL by gentle vortex. (2/2)

23.4.

Apply magnetic field, then remove supernatant and discard. (2/2)

24.

Resuspend bead pellet in 30µL.

Note

3X SDS Sample Loading Buffer is prepared 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%

25.

Incubate sample at 70°C for 0h 5m 0s.

26.

Apply magnetic field to sample, then load supernatant on SDS-PAGE gel and electrophorese.

Immunoprecipitation using Protein A/G Magnetic Beads

Abstract

Before start

Steps

Cell Lysis

Immunoprecipitation

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