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|2256S||100 µl (10 immunoprecipitations)||$246.00.0|
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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 get our images
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 utilizing magnetic separation.
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 (#70024) for mouse IgG immunoprecipitation.
- Magnetic Separation Rack: (#7017) or (#14654).
- 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 (Highly Recommended)
A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein G Magnetic beads. Pre-clear enough lysate for test samples and isotype controls.
- Briefly vortex the stock tube to resuspend the magnetic beads.
Transfer 20 μl of bead slurry to a clean tube. Place the tube in a magnetic separation rack for 10-15 seconds.
Carefully remove the buffer once the solution is clear. Add 500 μl of 1X cell lysis buffer to the magnetic bead pellet, briefly vortex to wash the beads. Place tube back in magnetic separation rack. Remove buffer once solution is clear. Repeat washing step once more.
- Add 200 μl cell lysate to 20 μl of pre-washed magnetic beads.
IMPORTANT: The optimal lysate concentration will depend on the expression level of the protein of interest. A starting concentration between 250 μg/ml-1.0 mg/ml is recommended.
- Incubate with rotation for 20 minutes at room temperature.
- Separate the beads from the lysate using a magnetic separation rack, transfer the pre-cleared lysate to a clean tube, and discard the magnetic bead pellet.
- Proceed to immunoprecipitation section.
IMPORTANT: Pre-wash #70024 magnetic beads just prior to use:
IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 for rabbit monoclonal primary antibodies, and Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples.
- Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate. Incubate with rotation overnight at 4°C to form the immunocomplex.
- Pre-wash magnetic beads (see Cell Lysate Pre-Clearing section, steps 1 and 2).
- Transfer the lysate and antibody (immunocomplex) solution to the tube containing the pre-washed magnetic bead pellet.
- Incubate with rotation for 20 min at room temperature.
- Pellet beads using magnetic separation rack. Wash pellets 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 20-40 µl 3X SDS sample buffer, briefly vortex to mix, and briefly microcentrifuge to pellet the sample.
- Heat the sample to 95-100°C for 5 min.
- Pellet beads using magnetic separation rack. Transfer the supernatant to a new tube. The supernatant is the sample.
- Load the sample (15-30 µl) on SDS-PAGE.
- Analyze sample by western blot (see Western Immunoblotting Protocol).
NOTE: For proteins with molecular weights in the range of around 50 kDa, we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) as a secondary antibody to minimize interference produced by denatured heavy chains. For proteins with molecular weights in the range of around 25 kDa, Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) is recommended to minimize interference produced by denatured light chains.
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 gel.
posted December 2008
revised October 2017
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).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.