Mouse (E1D5H) mAb IgG3 Isotype Control #37988

Immunoprecipitation for Native Proteins

This protocol is intended for immunoprecipitation of native proteins utilizing Protein G agarose beads for subsequent 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 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 G Agarose Beads: (#37478).
5. 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.
6. 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)

1. Vortex to mix beads.
2. Add 10–30 μl of 50% Protein G agarose bead slurry to 200 μl cell lysate at 1 mg/ml.
3. Incubate with rotation at 4°C for 30–60 min.
4. Microcentrifuge for 10 min at 4°C. Transfer the supernatant to a fresh tube.
5. 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 XP^® Isotype Control #3900 for rabbit monoclonal primary antibodies, Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal IgG1 primary antibodies, Mouse (E5Y6Q) mAb IgG2a Isotype Control #61656 for mouse monoclonal IgG2a primary antibodies, Mouse (E7Q5L) mAb IgG2b Isotype Control #53484 for mouse monoclonal IgG2b primary antibodies, and Mouse (E1D5H) mAb 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 at 1 mg/ml. Incubate with rotation overnight at 4°C.
2. Add Protein G agarose (10–30 μl of 50% bead slurry). Incubate with rotation 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 sample analysis by western immunoblotting or kinase activity (section D).

D. Sample Analysis

Proceed to one of the following specific set of steps.

For Analysis by Western Immunoblotting

1. Resuspend the pellet with 20 μl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec at 14,000 x g.
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: 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) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#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) mAb (#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) mAb (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 and microcentrifuge for 1 min at 14,000 x g.
7. Load the sample (15–30 μl) on SDS-PAGE (4–20%).