Upstream / Downstream
Explore pathways related to this product.
CST Antibody Performance Guarantee
To Purchase # 6937S
|6937S||400 µl (40 immunoprecipitations)||$329.00|
|$ 0. 00|
Find answers on our FAQs page.
PTM information and tools available.
EGF Receptor (D38B1) XP® Rabbit mAb (Magnetic Bead Conjugate) #6937
Immunoprecipitation of HeLa cell lysates using EGF Receptor (D38B1) XP® Rabbit mAb (Magnetic Bead Conjugate) (lane 1) and Rabbit (DA1E) mAb IgG XP® Isotype Control (Magnetic Bead Conjugate) #8726 (lane 2). The western blot was probed using EGF Receptor (D38B1) XP® Rabbit mAb #4267.Learn more about how we got this image
Gallery: EGF Receptor (D38B1) XP® Rabbit mAb (Magnetic Bead Conjugate) #6937
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
- 6-Tube Magnetic Separation Rack: (#7017).
- 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.
- Wash pellet by using magnetic rack to pellet beads, then discard supernatant once completely clear and add 500 µl of 1X cell lysis buffer, vortex to resuspend and wash the beads, repeat 4 more times for a total of 5 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 by using magnetic rack to pellet beads, then discard supernatant once completely clear and add 500 Âµl of 1X kinase buffer, vortex to resuspend and wash the beads, repeat 1 more time for a total of 2 washes. Keep on ice during washes.
- 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: 408
EGF Receptor (D38B1) XP® Rabbit mAb (Magnetic Bead Conjugate) detects endogenous levels of total EGF receptor protein. The antibody does not cross-react with other proteins of the ErbB family.Species Reactivity: Human, Mouse, Monkey
Monoclonal antibody is produced by immunizing animals with a fusion protein containing the cytoplasmic domain of human EGF receptor.
This Cell Signaling Technology antibody is immobilized by the covalent reaction of hydrazinonicotinamide-modifed antibody with formylbenzamide-modified magnetic bead. EGF Receptor (D38B1) XP® Rabbit mAb (Magnetic Bead Conjugate) is useful for immunoprecipitation assays of EGF receptor protein. The unconjugated EGF Receptor (D38B1) XP® Rabbit mAb #4267 reacts with human, mouse, and monkey EGF Receptor protein. CST expects that EGF Receptor (D38B1) XP® Rabbit mAb (Magnetic Bead Conjugate) will also recognize EGF Receptor in these species.
The epidermal growth factor (EGF) receptor is a transmembrane tyrosine kinase that belongs to the HER/ErbB protein family. Ligand binding results in receptor dimerization, autophosphorylation, activation of downstream signaling, internalization, and lysosomal degradation (1,2). Phosphorylation of EGF receptor (EGFR) at Tyr845 in the kinase domain is implicated in stabilizing the activation loop, maintaining the active state enzyme, and providing a binding surface for substrate proteins (3,4). c-Src is involved in phosphorylation of EGFR at Tyr845 (5). The SH2 domain of PLCγ binds at phospho-Tyr992, resulting in activation of PLCγ-mediated downstream signaling (6). Phosphorylation of EGFR at Tyr1045 creates a major docking site for the adaptor protein c-Cbl, leading to receptor ubiquitination and degradation following EGFR activation (7,8). The GRB2 adaptor protein binds activated EGFR at phospho-Tyr1068 (9). A pair of phosphorylated EGFR residues (Tyr1148 and Tyr1173) provide a docking site for the Shc scaffold protein, with both sites involved in MAP kinase signaling activation (2). Phosphorylation of EGFR at specific serine and threonine residues attenuates EGFR kinase activity. EGFR carboxy-terminal residues Ser1046 and Ser1047 are phosphorylated by CaM kinase II; mutation of either of these serines results in upregulated EGFR tyrosine autophosphorylation (10).
Protein Specific References
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.