BM7000 EGFR antibody
see related secondary antibodiessee all 203 EGFR products
0.1 ml / US$ 515
NOVUS BIOLOGICALS
PO Box 802 Littleton, CO 80160, USA
E-Mail: novus@novus-biologicals.com
Homepage: http://www.novus-biologicals.com
PO Box 802 Littleton, CO 80160, USA
E-Mail: novus@novus-biologicals.com
Homepage: http://www.novus-biologicals.com
Quick Overview
Mouse anti EGFR H9B4
Synonyms
Epidermal Growth Factor Receptor, ERBB1, Receptor tyrosine-protein kinase ErbB-1
Product Description
Mouse anti EGFR H9B4, Presentation: Purified. Product is tested for Immunoprecipitation ( IP ), Western blot / Immunoblot ( WB )
Properties
| Applications | Immunoprecipitation ( IP ), Western blot / Immunoblot ( WB ) |
| Reactivity | Human ( Hu ) |
| Presentation | Purified |
| Host | Mouse |
| Isotype | IgG1 |
| Clone | H9B4 |
| Catalog Number | BM7000 |
| Swiss Prot. No. | P00533 |
| Quantity | 0.1 ml |
| Price | US$ 515 |
| Delivery | Worldwide |
| Manufacturer | Acris Antibodies GmbH |
| Datasheet | view  BM7000.pdf |
Datasheet Extract
| Background | Epidermal Growth Factor Receptor (EGFR, wild type = 170 kDa, vIII variant = 145 kDa), also known as ErbB-1, is a transmembrane tyrosine kinase that regulates a variety of biological responses ranging from mitogenesis to stress signaling. The EGFR consists of a large extracellular domain, a single transmembrane domain and a cytoplasmic domain that exhibits kinase activity. Upon binding of EGF to the extracellular domain, the receptor undergoes dimerization and becomes phosphorylated on several tyrosine residues within the cytoplasmic domain. This results in EGFR activation and increased tyrosine kinase activity toward a variety of intracellular substrates. |
| Immunogen | Cytoplasmic domain of purified EGFR protein from human epidermoid carcinoma cells Swiss Num.: P00533 |
| Format | State: Liquid IgG fraction Purification: Protein G chromatography BufferSystem: 10 mM phosphate buffered saline, pH 7.4 (+/- 0.1), 50% glycerol with 1.0 mg/mL BSA (IgG, protease free) as a carrier, 0.05% sodium azide as preservative |
| Applications | Western blot (1:1000).
Immunoprecipitation. Use human epidermoid carcinoma (A431) cells as positive control. |
| Specificity | This antibody detects EGFR.
Useful as a positive control and for measuring total EGFR protein levels to compare signals obtained with the EGFR phosphorylation site specific antibodies. Species: Human. |
| Storage | Store at 2 - 8 °C up to one week or (in aliquots) at -20 °C for longer. Centrifuge vial before opening. Avoid repeated freezing and thawing.
Shelf life: one year from despatch. |
| References | Sieg, D.J., et al. (2000) FAK integrates growth-factor and integrin signals to promote cell migration. Nat. Cell. Biol. 2(5):249-256.
Biscardi, J.S., et al. (1999) c-Src-mediated phosphorylation of the epidermal growth factor receptor on Tyr845 and Tyr1101 is associated with modulation of receptor function. J. Biol. Chem. 274(12):8335-8343. Hashimoto, A., et al. (1999) Shc regulates epidermal growth factor-induced activation of the JNK signaling pathway. J. Biol. Chem. 274(29):20139-20143. Barbier, A.J., et al. (1999) Transmodulation of epidermal growth factor receptor function by cyclic AMP-dependent protein kinase. J. Biol. Chem. 274(20):14067-14073. Poppleton, H.M., et al. (1999) Modulation of the protein tyrosine kinase activity and autophosphorylation of the epidermal growth factor receptor by its juxtamembrane region. Arch. Biochem. Biophys. 363(2):227-236. |
| Protocols | Western Blotting Procedure
1. Lyse approximately 10e7 cells in 0.5 mL of ice cold Cell Lysis Buffer (formulation provided below). This buffer, a modified RIPA buffer, is suitable for recovery of most proteins, including membrane receptors, cytoskeletal-associated proteins, and soluble proteins. Other cell lysis buffer formulations, such as Laemmli sample buffer and Triton-X 100 buffer, are also compatible with this procedure. Additional optimization of the cell stimulation protocol and cell lysis procedure may be required for each specific application. 2. Remove the cellular debris by centrifuging the lysates at 14,000 x g for 10 minutes. Alternatively, lysates may be ultracentrifugedat 100,000 x g for 30 minutes for greater clarification. 3. Carefully decant the clarified cell lysates into clean tubes and determine the protein concentration using a suitable method, such as the Bradford assay. Polypropylene tubes are recommended for storing cell lysates. 4. React an aliquot of the lysate with an equal volume of 2x Laemmli Sample Buffer (125 mM Tris, pH 6.8, 10% glycerol, 10% SDS, 0.006% bromophenol blue, and 130 mM dithiothreitol [DTT]) and boil the mixture for 90 seconds at 100°C. 5. Load 10-30 µg of the cell lysate into the wells of an appropriate single percentage or gradient minigel and resolve the proteins by SDS-PAGE. 6. In preparation for the Western transfer, cut a piece of PVDF membrane slightly larger than the gel. Soak the membrane in methanol for 1 minute, then rinse with ddH2O for 5 minutes. Alternatively, nitrocellulose may be used. 7. Soak the membrane, 2 pieces of Whatman paper, and Western apparatus sponges in transfer buffer (formulation provided below) for 2 minutes. 8. Assemble the gel and membrane into the sandwich apparatus. 9. Transfer the proteins at 140 mA for 60-90 minutes at room temperature. 10. Following the transfer, rinse the membrane with Tris buffered saline for 2 minutes. 11. Block the membrane with blocking buffer (formulation provided below) for one hour at room temperature or overnight at 4°C. 12. Incubate the blocked blot with primary antibody at a 1:1000 dilution in Tris buffered saline supplemented with 3% BSA and 0.1% Tween 20 overnight at 4°C or for two hours at room temperature. 13. Wash the blot with several changes of Tris buffered saline supplemented with 0.1% Tween 20. 14. Detect the antibody band using an appropriate secondary antibody, such as goat F(ab)2 anti-rabbit IgG alkaline phosphatase conjugate or goat F(ab)2 anti-rabbit IgG horseradish peroxidase conjugate in conjunction with your chemiluminescence reagents and instrumentation. Cell Lysis Buffer Formulation: 10 mM Tris, pH 7.4 100 mM NaCl 1 mM EDTA 1 mM EGTA 1 mM NaF 20 mM Na4P2O7 2 mM Na3VO4 0.1% SDS 0.5% sodium deoxycholate 1% Triton-X 100 10% glycerol 1 mM PMSF (made from a 0.3 M stock in DMSO) or 1 mM AEBSF (water soluble version of PMSF) 60 µg/mL aprotinin 10 µg/mL leupeptin 1 µg/mL pepstatin (alternatively, protease inhibitor cocktail such as Sigma Cat. # P2714 may be used) Transfer Buffer Formulation: 2.4 gm Tris base 14.2 gm glycine 200 mL methanol Q.S. to 1 liter, then add 1 mL 10% SDS. Cool to 4°C prior to use. Tris Buffered Saline Formulation: 20 mM Tris-HCl, pH 7.4 0.9% NaCl Blocking Buffer Formulation: 100 mL Tris buffered saline 5 gm BSA 0.1 mL Tween 20 |
| Pictures | Western Blot Analysis Extracts of A431 cells untreated (-EGF) or treated with 200 ng/mL EGF for 15 minutes (+EGF) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer overnight at 4 °C, then incubated with each of the EGFR antibodies at their recommended dilution for two hours at room temperature in a 3% BSA-TBST buffer. After washing, the membrane was incubated with goat F(ab')2 anti-rabbit IgG alkaline phosphatase (to detect the polyclonal phosphorylation site specific antibodies), or goat F(ab')2 anti-mouse IgG alkaline phosphatase (to detect the monoclonal total antibodies). Signals were detected using the Tropix WesternStar TM method. Results show upregulation of each EGFR phosphorylation site, demonstrating the specificity of each antibody. The pan antibodies show total levels of EGFR irrespective of phosphorylation state. |
14 Secondary Antibodies
| Catalog No. | Name / Host | Presentation | Reactivity | ||||
|---|---|---|---|---|---|---|---|
| R1253B | Mouse IgG (H&L) | ||||||
| Rabbit | Biotin | 2 mg / US$ 315 | |||||
| R1253F | Mouse IgG (H&L) | ||||||
| Rabbit | FITC | 2 mg / US$ 295 | |||||
| R1253T | Mouse IgG (H&L) | ||||||
| Rabbit | TRITC | 2 mg / US$ 295 | |||||
| R1253TR | Mouse IgG (H&L) | ||||||
| Rabbit | Texas Red | 2 mg / US$ 305 | |||||
| R1253HRP | Mouse IgG (H&L) | ||||||
| Rabbit | HRP | 2 mg / US$ 325 | |||||
| R1253AP | Mouse IgG (H&L) | ||||||
| Rabbit | AP | 1 mg / US$ 335 | |||||
| R1457C2 | Mouse IgG (H&L) multi-species ads. | ||||||
| Goat | Cy2 | 1 mg / US$ 455 | |||||
| R1457C3 | Mouse IgG (H&L) multi-species ads. | ||||||
| Goat | Cy3 | 1 mg / US$ 455 | |||||
| R1457C35 | Mouse IgG (H&L) multi-species ads. | ||||||
| Goat | Cy3.5 | 1 mg / US$ 455 | |||||
| R1457C5 | Mouse IgG (H&L) multi-species ads. | ||||||
| Goat | Cy5 | 1 mg / US$ 455 | |||||
| R1457C55 | Mouse IgG (H&L) multi-species ads. | ||||||
| Goat | Cy5.5 | 1 mg / US$ 455 | |||||
| R1405R | Mouse IgG (H&L) F(ab')2 Fragment ads. to Bov, Eq, Hu, Rb, Rt, Sh | ||||||
| Goat | PE | 1 ml / US$ 465 | |||||
| R1403R | Mouse IgG (H&L) F(ab')2 Fragment ads. to Hu serum proteins | ||||||
| Rabbit | PE | 1 ml / US$ 465 | |||||
| R1455R | Mouse IgG (H&L) F(ab')2 Fragment multi-species ads. | ||||||
| Donkey | PE | 1 ml / US$ 465 | |||||
Click here to see all secondary antibodies for 'BM7000 EGFR'.