Immunoprecipitation for Native Proteins

This protocol is intended for immunoprecipitation of native proteins for analysis by western blot or kinase activity utilizing Protein A 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 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 Magnetic Beads: #73778
5. Magnetic Separation Rack: #7017 or #14654
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 (Highly Recommended)

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

1. Briefly vortex the stock tube to resuspend the magnetic beads.

   IMPORTANT: Pre-wash #73778 magnetic beads just prior to use.

2. Transfer 20 µL of bead slurry to a clean tube. Place the tube in a magnetic separation rack for 10-15 sec.

   Carefully remove the buffer once the solution is clear. Add 500 µL of 1X cell lysis buffer to the magnetic bead pellet, briefly vortex to wash the beads. Place tube back in magnetic separation rack. Remove buffer once solution is clear. Repeat washing step once more.

3. Add 200 µL cell lysate to 20 µL of pre-washed magnetic beads.

   IMPORTANT: The optimal lysate concentration will depend on the expression level of the protein of interest. A starting concentration of 250 µg/mL to 1.0 mg/mL is recommended.

4. Incubate with rotation for 20 min at room temperature.
5. Separate the beads from the lysate using a magnetic separation rack, transfer the pre-cleared lysate to a clean tube, and discard the magnetic bead pellet.
6. Proceed to immunoprecipitation below.

Immunoprecipitation

IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAB IgG Isotype Control #3900 for rabbit monoclonal primary antibodies, Mouse (G3A1) Monoclonal Antibody IgG1 Isotype Control #5415 for mouse monoclonal IgG1 primary antibodies, Mouse (E5Y6Q) Monoclonal Antibody IgG2a Isotype Control #61656 for mouse monoclonal IgG2a primary antibodies, Mouse (E7Q5L) Monoclonal Antibody IgG2b Isotype Control #53484 for mouse monoclonal IgG2b primary antibodies, and Mouse (E1D5H) Monoclonal Antibody IgG3 Isotype Control #37988 for mouse monoclonal IgG3 primary antibodies. Isotype controls should be concentration-matched and run alongside the primary antibody samples.

1. Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µL cell lysate. Incubate with rotation overnight at 4°C. to form the immunocomplex.

   IMPORTANT: For proteins prone to degradation, such as high molecular weight proteins, a 2-4 hr incubation at 4°C may be optimal.

2. Pre-wash magnetic beads (see Cell Lysate Pre-Clearing section, steps 1 and 2).
3. Transfer the lysate and antibody (immunocomplex) solution to the tube containing the pre-washed magnetic bead pellet.
4. Incubate with rotation for 20 min at room temperature.
5. Pellet beads using magnetic separation rack. Wash pellets five times with 500 µL of 1X cell lysis buffer. Keep on ice between washes.
6. Proceed to sample analysis by western blotting or kinase activity (section D).

D. Sample Analysis

For Analysis by Western Blotting

1. Resuspend the pellet with 20-40 µL 3X SDS sample buffer, briefly vortex to mix, then briefly microcentrifuge to pellet the sample.
2. Heat the sample to 95-100°C for 5 min.
3. Pellet beads using magnetic separation rack. Transfer the supernatant to a new tube. The supernatant is the sample.
4. Analyze sample by western blot (see Western Blotting Protocol).

NOTE: When using primary antibodies produced in rabbit to to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) Monoclonal Antibody #45262 or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) Monoclonal Antibody #3678 (or HRP conjugate #5127) as a secondary antibody to minimize interference produced by denatured rabbit heavy chain. For proteins with a molecular weight in the range of 25 kDa, Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) Monoclonal Antibody #3678 (or HRP conjugate #5127) is recommended to minimize interference produced by denatured mouse heavy or light chain.

When using primary antibodies produced in mouse to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Rabbit Anti-Mouse IgG (Light Chain Specific) (D3V2A) Monoclonal Antibody (HRP Conjugate) #58802 as a secondary antibody to minimize interference produced by denatured mouse heavy chain.

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, then microcentrifuge for 1 min at 14,000 x g.
7. Load the sample (15-30 µL) on SDS-PAGE gel.

For Research Use Only. Not for Use in Diagnostic Procedures.