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|9863P||200 µl (10 immunoprecipitations)||$65.00.0|
|9863S||1 ml (50 immunoprecipitations)||$132.00.0|
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- Additional protein information
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Protein A Agarose Beads #9863
Gallery: Protein A Agarose Beads #9863
- Immunoprecipitation (IP) of COS-7 cell extracts using Ubc12 (D13D7) Rabbit mAb #5641 and Protein A Agarose Beads #9863. Western blot analysis was performed on the IP pellet (lane 1) and supernatant (lane 2) using Ubc12 (D13D7) Rabbit mAb #5641.
Immunoprecipitation for Native Proteins
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.
- 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 A Agarose Beads: Use Protein A (#9863) for rabbit 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 A 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 A 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 December 2008
revised November 2013
Vortex tube briefly to resuspend the beads. Add 20 μl of bead slurry to each immunoprecipitation (IP) reaction. For bead washing and subsequent elution of immunocomplexes, the beads can be separated from solution by a brief 1 minute centrifugation in a microcentrifuge at 6,000 rpm. Resuspend the beads in solution by gently vortexing or rocking the tube. Please follow CST's recommended IP protocol to perform IP followed by western blot.Storage: Supplied as a 50% slurry in 18.5% ethanol solution. This product is stable for 12 months when stored at 4°C.
Protein A Agarose Beads are an affinity matrix for the small-scale isolation of immunocomplexes from immunoprecipitations (IP assays). Protein A is covalently coupled to agarose beads. Protein A exhibits high affinity for subclasses of IgG from many species (including human, rabbit, mouse, rat, and sheep) and can be used for immunoprecipitation assays with these antibodies.
Bead Diameter: 45-165 micron per bead
Binding Capacity: 20 +/- 2 mg human IgG/ml
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.