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 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 1X cell lysis buffer to 9 ml dH2O, mix.

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

  3. 3X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading 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 (#9863, #8687) for rabbit IgG immunoprecipitation and Protein G (#8740) for mouse IgG immunoprecipitation.

    NOTE: Magnetic beads (#8687, #8740) require 6-Tube Magnetic Separation Rack (#7017).

  5. Immobilized Streptavidin (Bead Conjugate) (For biotinylated antibodies): (#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 regulator for 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) 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 or magnetic beads (for unconjugated primary antibodies) or 10 µl streptavidin beads (#3419; for biotinylated antibodies), to 200 µl cell lysate at 1 mg/ml.
  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.

Using Unconjugated Primary Antibodies

  1. 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 or magnetic beads (10–30 µl of 50% bead slurry). Incubate with gentle rocking for 1–3 hr at 4°C for agarose beads, or 10–30 min for magnetic beads.
  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 in the product 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 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 during washes.
  4. Proceed to analyze by western immunoblotting or kinase activity (Section D).

Using Immobilized Antibodies (Sepharose® Bead Conjugate)

  1. 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 by western immunoblotting or kinase activity (Section D).

Using Immobilized Antibodies (Magnetic Bead Conjugate)

  1. 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 (#7017) 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 by western immunoblotting or kinase activity (Section D).

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 a 4–20% gel for SDS-PAGE.
  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%).

posted December 2008

revised November 2013

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