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EGF Receptor (EGFR1) Mouse mAb (IP Specific) #2256
Western blot analysis of EGF Receptor (EGFR1) Mouse mAb (IP Specific) immunoprecipitated samples from Gefitinib-treated and untreated HCC827 cell lysates, using Phospho-EGF Receptor (Tyr1068) Antibody (#2234) (upper) and EGF Receptor Antibody (#2232) (lower).Learn more about how we got this image
Gallery: EGF Receptor (EGFR1) Mouse mAb (IP Specific) #2256
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 G Magnetic Beads: Use Protein G (#8740) for mouse IgG immunoprecipitation.
- 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 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 magnetic bead slurry to 200 µl cell lysate at 1 mg/ml.
- Incubate with rotation at 4°C for 30–60 min.
- Pellet beads using magnetic separation rack. 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 magnetic beads (10–30 µl of 50% bead slurry). Incubate with rotation for 10–30 min at 4°C.
- Pellet beads using magnetic separation rack. 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).
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).
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
protocol id: 121
EGF Receptor (EGFR1) Mouse mAb (IP Specific) specifically immunoprecipitates endogenous EGF receptors from various cell lysates. This antibody does not cross-react with other EGF receptor family members.Species Reactivity: Human
Monoclonal antibody is produced by immunizing animals with a recombinant protein corresponding to the extracellular domain of human EGF receptor.
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.