Immunoprecipitation for Native Proteins
#9356 Phospho-Stat5 (Tyr694) (14H2) Mouse mAb用プロトコール
This protocol is intended for immunoprecipitation of native proteins using Mouse IgG with 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.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L of 1X PBS, add 50 ml 20X PBS to 950 ml dH2O, mix.
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.
- 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.
- Protein G Agarose Beads: Use Protein G (#37478) for Mouse IgG 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.
Cell Lysate Pre-Clearing (Optional)
- Vortex to mix beads.
- Add 10–30 μl of 50% Protein G agarose bead slurry to 200 μl cell lysate at 1 mg/ml.
- 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 immunoprecipitation below.
- 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.
- Add Protein G agarose (10–30 μl of 50% bead slurry). Incubate with rotation 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 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
- Resuspend the pellet with 20 μl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec at 14,000 x g.
- 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 a 4–20% gel for SDS-PAGE.
- Analyze sample by western blot (see Western Immunoblotting Protocol).
NOTE: To minimize masking caused by denatured IgG heavy chains (~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) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
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 (4–20%).
posted October 2016