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Jak2 (D2E12) XP® Rabbit mAb (Sepharose Bead Conjugate) #4089
Immunoprecipitation of extracts from K562 cells, using Jak2 (D2E12) XP® Rabbit mAb (Sepharose Bead Conjugate) (lane 2). XP® Rabbit IgG (DA1E) mAb Isotype Control (Sepharose Bead Conjugate) #3423 was used as a negative control (lane 1). The western blot was probed using Jak2 (D2E12) XP® Rabbit mAb #3230 and detected with Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb #3678 followed by Anti-mouse IgG, HRP-linked Antibody #7076.Learn more about how we got this image
Gallery: Jak2 (D2E12) XP® Rabbit mAb (Sepharose Bead Conjugate) #4089
Immunoprecipitation for Analysis by Western Blotting
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 equivalently purified water.
- 20X Phosphate Buffered Saline (PBS): (#9808).
10X Cell Lysis Buffer: (#9803) 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml Leupeptin
NOTE: CST recommends adding 1 mM PMSF (#8553) before use*.
- 3X SDS Sample Buffer: (#7722) 187.5 mM Tris-HCl (pH 6.8 at 25°C), 6% w/v SDS, 30% glycerol, 150 mM DTT, 0.03% w/v bromophenol blue
- 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 PBS.
- Remove PBS and add 0.5 ml 1X ice-cold cell lysis buffer to each plate (10 cm) and incubate the plates on ice for 5 minutes.
- Scrape cells off the plates and transfer to microcentrifuge tubes. Keep on ice.
- Sonicate samples on ice three times for 5 seconds each.
- Microcentrifuge for 10 minutes at 4°C, 14,000 x g, and transfer the supernatant to a new tube. If necessary, lysate can be stored at –80°C.
- Take 200 μl cell lysate and add 10 μl of the immobilized antibody, incubate with rotation overnight at 4°C.
- Microcentrifuge for 30 seconds at 4°C. Wash pellet five times with 500 μl of 1X cell lysis buffer. Keep on ice during washes.
- Proceed to sample analysis by western blotting 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 2007
protocol id: 27
Jak2 (D2E12) XP® Rabbit mAb (Sepharose Bead Conjugate) detects endogenous levels of total Jak2 protein. No cross-reactivity was observed with other family members.Species Reactivity: Human, Mouse, Rat Species predicted to react based on 100% sequence homology: Hamster, Monkey, Chicken, Xenopus, Bovine, Dog, Pig, Horse
Monoclonal antibody is produced by immunzing animals with a synthetic peptide corresponding to residues surrounding Pro841 of Jak2.
This Cell Signaling Technology antibody is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated sepharose beads. Jak2 (D2E12) XP® Rabbit mAb (Sepharose Bead Conjugate) is useful for the immunoprecipitation of Jak2 proteins.
Members of the Janus family of tyrosine kinases (Jak1, Jak2, Jak3, and Tyk2) are activated by ligands binding to a number of associated cytokine receptors (1). Upon cytokine receptor activation, Jak proteins become autophosphorylated and phosphorylate their associated receptors to provide multiple binding sites for signaling proteins. These associated signaling proteins, such as Stats (2), Shc (3), insulin receptor substrates (4), and focal adhesion kinase (FAK) (5), typically contain SH2 or other phospho-tyrosine-binding domains.
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc. XP® is a trademark of Cell Signaling Technology, Inc. Jak antibodies produced under license (granting certain rights including those under U.S. Patent No. 5,658,791) from Chemicon International, Inc.