EGFR antibody (Epidermal Growth Factor Receptor)

Details for Product anti-EGFR Antibody No. ABIN98862
Antigen
Reactivity
Human
2045
824
766
31
23
20
18
16
14
6
4
3
2
2
2
2
2
2
1
1
1
1
Host
Rabbit
1446
552
23
19
13
5
2
1
1
1
Clonality
Polyclonal
Conjugate
This EGFR antibody is un-conjugated
101
53
44
43
41
41
41
41
41
35
26
22
16
16
16
16
16
8
8
8
8
8
8
8
8
8
8
8
8
8
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
1
Application
Immunoprecipitation (IP), ELISA, Western Blotting (WB)
1288
575
508
339
301
272
212
210
74
67
66
56
32
31
19
19
14
11
9
6
6
4
4
3
3
3
3
3
2
2
2
1
1
1
1
1
1
1
1
1
Options
'Independent Validation' Badge
Lot Number 19538
Method validated Western Blotting
Positive Control A549 cells
Negative Control MCF-7 cells
Notes A strong specific band was observed in the positive control at the expected size (~175 kDa) that is not observed in the negative control.
Primary Antibody
  • Antigen: Epidermal Growth Factor Receptor (EGFR)
  • Catalog number: ABIN98862
  • Lot number: 19538
  • Dilution: 1:1,000
Secondary Antibody
  • Antibody: IRDye 680LT Goat Anti-Rabbit
  • Lot number: C30725-01
  • Dilution: 1:10,000
Controls - Lysates were prepared by ADS Biosystems following standard protocols and quality controlled for protein integrity on a regular basis
Protocol
  • Lysates were mixed with NuPAGE® LDS Sample Buffer (Life Technologies NP0007) and NuPAGE® Sample Reducing Agent (Life Technologies NP0004) and denatured for 5 minutes at 90ºC.
  • 40 μg of each lysate was electrophoresed on a Bolt 4-12% Bis-Tris Gel (Life Technologies BG04120BOX) and run in Bolt MOPS SDS Running Buffer (Life Technologies B0001) at 160 volts for 1 hour.
  • Odyssey Western Protein Standard (LI-COR #928-40000) was run as a molecular weight standard.
  • PVDF membrane was activated with methanol.
  • Protein samples were transferred to activated PVDF membrane in a wet Bolt Transfer Apparatus (Life Technologies B1000) at room temperature for 1 hour at 20 volts (started at 230mA, ended at 110mA).
  • The membrane was blocked in x LI-COR Odyssey WB block solution for 1 hour at room temperature.
  • The membrane was incubated with the primary antibody diluted 1:1000 in x LI-COR Odyssey WB block solution incubated 2 hours at room temperature.
  • The membrane was washed 4 x 5 minutes in 1 x PBS-T (PBS solution with 0.1% Tween 20).
  • The membrane was incubated with IRDye® 800CW Goat anti-Mouse Secondary Antibody (Red) and IRDye 680LT Goat Anti-Rabbit Secondary Antibody (Green) from LI-COR (#827-11081, Lot #C30725-01), both 1:10,000 dilutions. Incubation was performed at room temperature for 45 minutes.
  • The membrane was washed 4 x 5 minutes in 1 x PBS-T (PBS solution with 0.1% Tween 20).
  • Proteins were detected using Odyssey machine scanning with green channel for loading control and red channel for potential LPL band.
Experimental Notes - No experimental challenges noted.
Validation Images
Western Blotting validation image for anti-Epidermal Growth Factor Receptor (EGFR) antibody (ABIN98862) Figure 1: Scanned image of EGFR (Green) and loading control alpha-tubulin (Red) Weste...
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.
Characteristics Concentration Definition: by Refractometry
Antigen
Alternative Name EGFR (EGFR Antibody Abstract)
Background EGFR is a transmembrane glycoprotein that is a member of a family of protein tyrosine kinases crucial to maintaining a normal balance in cell growth and development.  Growth factor receptors are involved not only in promoting the proliferation of normal cells but also in the aberrant growth of many types of human tumors.  For example, the epidermal growth factor receptor (EGFR) is mutated and/or over-expressed in many common solid human squamous cell carcinomas including breast, brain, bladder, lung, gastric, head & neck, esophagus, cervix, vulva, ovary, and endometrium. Over-expression of the EGFR gene occurs in carcinomas with and without gene amplification. EGFR and ErbB-2 are particularly important in breast cancer because increased production or activation has been associated with poor prognosis. EGFR belongs to a family of growth factor receptors, which also includes ErbB-2/HER-2/neu, ErbB-3/HER-3/neu and ErbB-4/HER-4/neu. EGFR can heterodimerize with each of the members of this family.
Synonyms: Receptor tyrosine-protein kinase erbB-1 antibody, c-erbB-1 antibody
Gene ID 1956, 29725609
UniProt P00533
Pathways NF-kappaB Signaling, RTK Signaling, Fc-epsilon Receptor Signaling Pathway, EGFR Signaling Pathway, Neurotrophin Signaling Pathway, Stem Cell Maintenance, Hepatitis C, Positive Regulation of Response to DNA Damage Stimulus, Interaction of EGFR with phospholipase C-gamma, Thromboxane A2 Receptor Signaling, EGFR Downregulation
Application Notes Anti-EGFR antibody is specifically designed for ELISA, immunoblotting and immunoprecipitation.  Reactivity in other assays is likely, but has not been determined.  Recognition of EGFR is independent of the phosphorylation status at tyrosine 1173.  No reaction is observed against ErbB-2, ErbB-3 or ErbB-4.   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.
Restrictions For Research Use only
Format Liquid
Concentration 85 mg/mL
Preservative Sodium azide
Precaution of Use This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Storage -20 °C
Supplier Images
Image no. 1 for anti-Epidermal Growth Factor Receptor (EGFR) antibody (ABIN98862) Combined immunoprecipitation and western blot using anti-EGFR antibody. Lysates were ...
Image no. 2 for anti-Epidermal Growth Factor Receptor (EGFR) antibody (ABIN98862) Combined immunoprecipitation and western blot using anti-EGFR antibody. Lysates were ...
Product cited in: Andersson, Anttonen, Färkkilä, Pihlajoki, Bützow, Unkila-Kallio, Heikinheimo: "Sensitivity of human granulosa cell tumor cells to epidermal growth factor receptor inhibition." in: Journal of molecular endocrinology, Vol. 52, Issue 2, pp. 223-34, 2014 (PubMed).

Khoh-Reiter, Jessen: "Evaluation of the cytotoxic effects of ophthalmic solutions containing benzalkonium chloride on corneal epithelium using an organotypic 3-D model." in: BMC ophthalmology, Vol. 9, pp. 5, 2009 (PubMed).

Miettinen, Ustinov, Ormio, Gao, Palgi, Hakonen, Juntti-Berggren, Berggren, Otonkoski: "Downregulation of EGF receptor signaling in pancreatic islets causes diabetes due to impaired postnatal beta-cell growth." in: Diabetes, Vol. 55, Issue 12, pp. 3299-308, 2007 (PubMed).