EGFR Antibody

SKU: orb750443

Description

EGFR antibody

Research Area

Cell Biology

Images & Validation

• Tested Applications: ELISA, IHC, IP, WB
• Dilution Range: ELISA: 1:10,000 - 1:50,000, IHC: 2.5 µg/mL, IP: 10 µl, WB: 1:1,000 - 1:10,000
• Reactivity: Human, Rat
• Application Notes:
  Anti-EGFR antibody has been tested by and is specifically designed for ELISA, immunoblotting, immunoprecipitation, and immunohistochemistry. Reactivity in other assays is likely, but has not been determined. Recognition of EGFR is independent of the phosphorylation status at tyrosine 1173. A431 cells, keratinocytes in normal epidermis, or placenta are typically used as positive control sources. The antigen is typically localized in the cell membrane. For western blotting, good results are also achieved on PVDF membranes blocked with 5% lowfat milk diluted in TTBS for 1 hour at room temperature. Also, dilute the primary antibody and secondary in 5% lowfat milk in TTBS. Anti-EGFR can be diluted up to 1:10,000 for immunoblot depending on the cell line and the amount of EGFR in a particular lysate. For immunoprecipitation, use approximately 10 µl of the antibody. The immunoprecipitation mix should contain the antibody, 25 µl of Protein A-agarose beads and 1.0 ml of lysate (lysate contains approximately 1.0 mg of total protein). This mixture should be rotated overnight at 4°C and then washed 3 times with lysis buffer (used to prepare the lysate). The resulting bead complex is dissolved in 20-30 µl of 3X SDS-PAGE sample buffer and approximately 15 µl is loaded per lane on an 8% polyacrylamide gel.

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Key Properties

• Antibody Type: Primary Antibody
• Host: Rabbit
• Clonality: Polyclonal
• Isotype: Antiserum
• Immunogen: This whole rabbit serum was prepared by repeated immunizations with a peptide synthesized using conventional technology. The sequence of the epitope maps to a region near the carboxy terminus which is identical in human, mouse and rat EGFR.
• Target: EGFR
• Purity: This antiserum is directed against human epidermal growth factor receptor (EGFR) and is useful in determining its presence in western blotting and immunoprecipitation experiments. This antibody can detect EGFR from human, mouse and rat sources. Reactivity of this antibody with EGFR from other species is unknown. No reaction is observed against ErbB-2, ErbB-3 or ErbB-4.
• Conjugation: Unconjugated

Storage & Handling

• Storage: Store vial at -20° C prior to opening. Aliquot contents and freeze at -20° C or below for extended storage. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4° C as an undiluted liquid. Dilute only prior to immediate use.
• Form/Appearance: Liquid (sterile filtered)
• Buffer/Preservatives: 0.01% (w/v) Sodium Azide
• Concentration: 85 mg/mL
• Expiration Date: 12 months from date of receipt.
• Dry Ice Shipping: Please note: This product requires shipment on dry ice. A dry ice surcharge will apply.
• Disclaimer: For research use only

Alternative Names

rabbit anti-EGFR Antibody, rabbit anti-epidermal growth factor receptor antibody, Receptor tyrosine-protein kinase erbB-1 antibody, c-erbB-1 antibody

Combined immunoprecipitation and western blot using anti-EGFR antibody. Lysates were prepared from GN4 rat liver epithelial cells both with (+) EGF treatment for 15' at 100 ng/mL and without (-) the addition of EGF. The combination of immunoprecipitation and western blotting was performed using the anti-EGFR antibody for immunoprecipitation (10 µl) followed by western blot detection using an anti-phosphotyrosine antibody (Panel A). This was repeated in reverse order using a 1:2000 dilution of anti-EGFR for western blot (Panel B). Visualization occurred using an ECL system. Film exposure was approximately 1'. Other detection systems will yield similar results.

Immunohistochemistry of Anti-EGFR Antibody with positive staining.

Western blot using Biorbyt's anti-EGFR antibody. Lane 1: unstimulated A431 whole cell lysates (p/n orb348665). Lane 2: EGF stimulated A431 whole cell lysates. Shows detection of a band at ~170 kDa corresponding to human EGFR present in unstimulated and stimulated (50 ng/mL for 15 min) lysates (arrowhead). Loaded: 30 µg lysate was resolved on a 4-20% Tris-Glycine gel by SDS-PAGE and transferred onto nitrocellulose. Primary Antibody: Anti-EGFR at 1:1000 overnight at 4°C. Secondary Antibody: IRDye® 800 conjugated Gt-a-Rabbit IgG (H&L) MX10 at 1:10000 dilution of for 45 min at room temperature (800 nm channel, green). Molecular weight estimation was made by comparison to prestained MW markers in lane M (700 nm channel, red).

Quick Database Links

Gene Symbol

EGFR

UniProt

P00533

RefSeq:29725609