This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity. Please refer to our Immunoprecipitation Protocol Utilizing Magnetic Separation when using our #73778 Protein A Magnetic Beads or #70024 Protein G Magnetic Beads.
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L of 1X PBS, add 50 ml 20X PBS to 950 ml dH2O, mix.
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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.
- 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.
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Protein A or G Agarose Beads (For unconjugated primary antibodies): Use Protein A (#9863) for rabbit IgG immunoprecipitation and Protein G (#37478) for mouse IgG immunoprecipitation.
- Immobilized Streptavidin (Bead Conjugate) (For biotinylated antibodies): (#3419) Gently vortex vial and use 10 µl per immunoprecipitation.
- 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.
- 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
- Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
- To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.
- 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.
- Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice.
- Sonicate on ice three times for 5 sec each.
- 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.)
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.
- To 200 μl cell lysate at 1 mg/ml, add 20 μl of Protein A (#9863) or Protein G (#37478) agarose 50% bead slurry, 10 μl Streptavidin (Sepharose® Bead Conjugate #3419) for biotinylated antibodies.
- Incubate with rotation at 4°C for 30-60 min.
- Microcentrifuge for 10 min at 4°C. Transfer the supernatant to a fresh tube.
- Proceed to one of the following specific set of steps depending on the primary antibody used.
Using Unconjugated Primary Antibodies
- Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate. Incubate with gentle rocking overnight at 4°C.
- Add either Protein A (#9863) or Protein G (#37478) agarose (20 µl of 50% bead slurry). Incubate with gentle rocking for 1-3 hr at 4°C.
- Microcentrifuge for 30 sec at 4°C. Wash pellet five times with 500 µl of 1X cell lysis buffer. Keep on ice between washes.
- Proceed to analyze by western immunoblotting or kinase activity (Section D).
Using Biotinylated Primary Antibodies
- Add biotinylated antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate. Incubate with gentle rocking overnight at 4°C.
- Gently mix Immobilized Streptavidin (Sepharose® Bead Conjugate #3419) and add 10 µl of slurry. Incubate with gentle rocking for 2 hr at 4°C.
- Microcentrifuge for 30 sec at 4°C. Wash pellet five times with 500 µl of 1X cell lysis buffer. Keep on ice during washes.
- Proceed to analyze by western immunoblotting or kinase activity (Section D).
Using Immobilized Antibodies (Sepharose® Bead Conjugate)
- 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.
- Microcentrifuge for 30 sec at 4°C. Wash pellet five times with 500 µl of 1X cell lysis buffer. Keep on ice during washes.
- Proceed to analyze by western immunoblotting or kinase activity (Section D).
Using Immobilized Antibodies (Magnetic Bead Conjugate)
- 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.
- Pellet magnetic beads by placing the tubes in a magnetic separation rack (#7017 or #14654) 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.
- 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 or #14654).
For Analysis by Western Immunoblotting
- Resuspend the pellet with 20-40 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
- Heat the sample to 95-100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
- Load the sample (15-30 µl) on SDS-PAGE gel.
- 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) (D4W3E) mAb (#45262) 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
- Wash pellet twice with 500 µl 1X kinase buffer. Keep on ice.
- Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate.
- Incubate for 30 min at 30°C.
- Terminate reaction with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
- Transfer supernatant containing phosphorylated substrate to another tube.
- Heat the sample to 95-100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
- Load the sample (15-30 µl) on SDS-PAGE gel.
posted December 2008
revised April 2021
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