Immunoprecipitation (For Native Protein)

This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.

A. Solutions and Reagents

NOTE: Prepare solutions with purified 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 1 L of 1X Cell Lysis Buffer, add 100 ml Cell Lysis Buffer to 900 ml dH₂O, mix. NOTE: Add 1 mM PMSF immediately prior to use.
3. Blue Loading Buffer Pack (SDS loading buffer): (#7722) Prepare fresh 3X reducing SDS loading buffer by adding 1/10 volume 30X reducing agent (1.25 M) to 1 volume of 3X SDS loading buffer.
4. Protein A or G Agarose Beads (For unconjugated primary antibodies): (Protein A #9863) Please prepare according to manufacture’s instructions. Use Protein A for rabbit IgG pull down and Protein G for mouse IgG pull down.
5. Immobilized Streptavidin (Bead Conjugate) (For biotinylated antibodies): (#3419) Gently vortex vial and use 10 µl per pull down.
6. 10X Kinase Buffer: (#9802) To Prepare 1 ml of 1X Kinase Buffer, add 100 µl 10X Kinase Buffer to 900 µl dH₂O, mix.
7. ATP (10 mM): (#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 modulator for desired time.
2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold PBS.
3. Remove PBS and add 0.5 ml 1X ice-cold cell lysis buffer to each plate (10 cm) and incubate on ice for 5 min.
4. Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice.
5. Sonicate on ice three times for 5 sec each.
6. Microcentrifuge for 10 min at 4°C, 14,000 X g and 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.)

1. Add 10–30 µl of 50% bead slurry, either Protein A or G agarose beads (for unconjugated primary antibodies) or 10 µl streptavidin beads (for biotinylated antibodies) to 200 µl cell lysate at 1 mg/ml.
2. Incubate at 4°C for 30–60 min.
3. Spin 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.

Using Unconjugated Primary Antibodies

1. Microcentrifuge lysate for 1 min at 14,000 X g. Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate at 1 mg/ml. Incubate with gentle rocking overnight at 4°C.
2. Add either protein A or G agarose beads (10–30 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 during washes.
4. Proceed to analyze pellet by western immunoblotting or kinase activity (Section D).

Using Biotinylated Primary Antibodies

1. Microcentrifuge lysate for 1 min at 14,000 X g. Add biotinylated antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate at 1 mg/ml. Incubate with gentle rocking overnight at 4°C.
2. Vortex gently and add Immobilized Streptavidin (Bead Conjugate) (#3419) (10 µl). 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 during washes.
4. Proceed to analyze pellet by western immunoblotting or kinase activity (see below).

Using Immobilized Antibodies (Sepharose Bead Conjugate)

1. Microcentrifuge lysate for 1 min at 14,000 X g. Vortex gently 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 during washes.
3. Proceed to analyze pellet by western immunoblotting or kinase activity (Section D).

Using Immobilized Antibodies (Magnetic Bead Conjugate)

1. Microcentrifuge lysate for 1 min at 14,000 X g. Vortex gently 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 and wait 1 to 2 min for solution to clear. Wash pellet five times with 500 µl of 1X cell lysis buffer. Keep on ice during washes.
3. Proceed to analyze pellet by western immunoblotting or kinase activity (see below).

D. Sample Analysis

Proceed to one of the following specific set of steps. NOTE: For magnetic beads, do not centrifuge. Instead use a magnetic separation rack (#7017).

For Analysis by Western Immunoblotting

1. Resuspend the pellet with 20 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 (4–20%).
4. Analyze sample by western blot (see Western Immunoblotting Protocol).

NOTE: For proteins with molecular weights near 50 kDa, we recommend using Mouse Anti-rabbit IgG (Light-Chain Specific) (L57A3) mAb #3677 or Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 as a secondary antibody to minimize masking produced by denatured heavy chains. For proteins with molecular weights near 25 kDa, Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 or Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 is recommended.

For Analysis by Kinase Assay

1. Wash pellet twice with 500 µl 1X kinase buffer. Keep on ice.
2. Suspend 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 and microcentrifuge for 1 min at 14,000 X g.
7. Load the sample (15–30 µl) on SDS-PAGE (4–20%).