Immunoprecipitation Protocol (For Native Protein)

This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblotting or kinase activity. When using Protein A Magnetic Beads #70024, please refer to the Immunoprecipitation Protocol Utilizing Magnetic Separation.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. 20X Phosphate Buffered Saline (PBS) #9808: To prepare 1 L of 1X PBS, add 50 mL 20X PBS to 950 mL dH2O, mix.
2. 10X Cell Lysis Buffer #9803: To prepare 10 mL of 1X cell lysis buffer, add 1 mL 10X cell lysis buffer to 9 mL dH2O, mix.

   NOTE: Add 1 mM PMSF #8553 immediately prior to use.

3. 3X SDS Sample Buffer: Use Blue Loading Buffer Pack #7723. Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer.
4. Protein A or G Agarose Beads (For unconjugated primary antibodies): Use Protein A #37478 for mouse IgG immunoprecipitation.
5. Immobilized Streptavidin (Sepharose Bead Conjugate) #3419: Gently vortex vial and use 10 µL per immunoprecipitation.
6. 10X Kinase Buffer (For kinase assays) #9802: To prepare 1 mL of 1X kinase buffer, add 100 µL 10X kinase buffer to 900 µL dH2O, mix.
7. ATP (10 mM) (For kinase assays) #9804: To prepare 0.5 mL of ATP (200 µM), add 10 µL ATP (10 mM) to 490 µL 1X kinase buffer.

B. Preparing Cell Lysates

1. Aspirate media. Treat cells by adding fresh media containing a regulator for the desired time.
2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.
3. Remove PBS and add 0.5 mL ice-cold 1X cell lysis buffer to each plate (10 cm), then incubate the plates on ice for 5 min.
4. Scrape cells off the plates and transfer to microcentrifuge tubes. Keep on ice.
5. Sonicate samples on ice three times for 5 sec each.
6. Microcentrifuge for 10 min at 4°C, 14,000 x g, then transfer the supernatant to a new tube. The supernatant is the cell lysate. If necessary, lysate can be stored at -80°C.

C. Immunoprecipitation

Cell Lysate Pre-Clearing (Optional Step for Unconjugated and Biotinylated Antibodies)

A cell lysate pre-clearing step is highly recommended to reduce nonspecific protein binding to the Protein A, Protein G, or Streptavidin beads. Pre-clear enough lysate for test samples and isotype controls.

1. To 200 µL cell lysate at 1 mg/mL, add 20 µL of Protein A
2. Incubate with rotation at 4°C for 30-60 min.
3. Microcentrifuge for 10 min at 4°C. Transfer the supernatant to a fresh tube.
4. Proceed to one of the following specific set of steps depending on the primary antibody used.

Immunoprecipitation

IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Isotype controls should be concentration-matched and run alongside the primary antibody samples.

Using Unconjugated Primary Antibodies

1. Add primary antibody at the appropriate dilution, as recommended on the product webpage or datasheet, to 200 µL cell lysate at 1 mg/mL. Incubate with gentle rocking overnight at 4°C.
2. Add either Protein A #37478 agarose (20 µL of 50% bead slurry). Incubate with gentle rocking for 1-3 hr at 4°C.
3. Microcentrifuge for 30 sec at 4°C. Wash pellet five times with 500 µL of 1X cell lysis buffer. Keep on ice between washes.
4. Proceed to analyze by western immunoblotting or kinase activity (Section D).

Using Biotinylated Primary Antibodies

1. Add biotinylated antibody at the appropriate dilution, as recommended on the product webpage or datasheet, to 200 µL cell lysate at 1 mg/mL. Incubate with gentle rocking overnight at 4°C.
2. Gently mix immobilized Streptavidin (Sepharose Bead Conjugate) #3419 and add 10 µL of bead slurry. Incubate with gentle rocking for 2 hr at 4°C.
3. Microcentrifuge for 30 sec at 4°C. Wash pellet five times with 500 µL of 1X cell lysis buffer. Keep on ice between washes.
4. Proceed to analyze by western immunoblotting or kinase activity (Section D).

Using Immobilized Antibodies (Sepharose Bead Conjugate)

1. Gently mix and add immobilized bead conjugate (10 µL) to 200 µL cell lysate at 1 mg/mL. Incubate with gentle rocking overnight at 4°C.
2. Microcentrifuge for 30 sec at 4°C. Wash pellet five times with 500 µL of 1X cell lysis buffer. Keep on ice between washes.
3. Proceed to analyze by western immunoblotting or kinase activity (Section D).

Using Immobilized Antibodies (Magnetic Bead Conjugate)

1. Gently mix and add immobilized bead conjugate (10 µL) to 200 µL cell lysate at 1 mg/mL. Incubate with gentle rocking overnight at 4°C.
2. Pellet magnetic beads by placing the tubes in a magnetic separation rack #14654.
3. Proceed to analyze by western immunoblotting or kinase activity (Section D).

D. Sample Analysis

Proceed to one of the following specific sets of steps depending on the analysis type.

NOTE: For magnetic bead conjugates, do not centrifuge; instead use a magnetic separation rack #14654.

For Analysis by Western Immunoblotting

1. Resuspend the pellet with 20-40 µL 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
2. Heat the sample to 95-100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
3. Load the sample (15-30 µL) on SDS-PAGE gel.
4. Analyze the sample by western immunoblotting. Please refer to the Western Immunoblotting Protocol.

NOTE: to minimize masking of target bands produced by denatured heavy chains or interference produced by denatured light chains, the following secondaries are suggested:

For proteins with molecular weights at approximately 50 kDa:

Mouse Anti-rabbit IgG (Light-Chain Specific) (D4W3E) mAb #45262

Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (HRP Conjugate) #93702

Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678

Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127

For proteins with molecular weights at approximately 25 kDa:

Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678

Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127

For Analysis by Kinase Assay

1. Wash the pellet twice with 500 µL 1X kinase buffer. Keep on ice.
2. Resuspend the pellet in 40 µL 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate.
3. Incubate for 30 min at 30°C.
4. Terminate reaction with 20 µL 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
5. Transfer supernatant containing phosphorylated substrate to another tube.
6. Heat the sample to 95-100°C for 2-5 min, then microcentrifuge for 1 min at 14,000 x g.
7. Load the sample (15-30 µL) on SDS-PAGE gel.