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You are here:Home » Antibodies » Abs to Oncoproteins » Anti -ErbB2, phosphorylated, Tyr1248 (c-erbB2, HER2, neu Oncoprotein)

Anti -ErbB2, phosphorylated, Tyr1248 (c-erbB2, HER2, neu Oncoprotein)

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Specifications

Clone Host Grade Applications
Polyclonal Rabbit Affinity Purified B
Catalog #E3451-25
ApplicationsSuitable for use in Western Blot. Other applications not tested.
Recommended DilutionWestern Blot: 1:2000 -1:8000 dilutions detects activated Erb B2/HER-2 in RIPA lysates from heregulin stimulated MCF-7 breast carcinoma cells. Lysates from non-stimulated and heregulin-stimulated MCF-7 cells were resolved by electrophoresis, transferred to nitrocellulose and probed with E3451-25 (1:2000 dilution). Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system.
Optimal dilution determined by the researcher.
Immunoblot Protocol 1. Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a cell lysate sample (cell lysis buffer 50mM Tris-HCl, pH7.4; 1% NP-40; 0.25% sodium deoxycholate; 150mM NaCl; 1mM EGTA; 1mM PMSF; 1ug/ml each aprotinin, leupeptin, pepstatin; 1mM Na3VO4; 1mM NaF) and transfer the proteins to nitrocellulose. Wash the blotted nitrocellulose twice with water. 2. Block the blotted nitrocellulose in freshly prepared PBS containing 3% nonfat dry milk (PBS-MLK) for 30 minutes at RT with constant agitation. 3. Incubate the nitrocellulose with a 1:2000-1:8000 dilution of E3451-25 diluted in freshly prepared PBS-MLK overnight with agitation at 4°C. 4. Wash the nitrocellulose twice with water. 5. Incubate the nitrocellulose in the secondary reagent of choice (a goat anti-rabbit HRP conjugated IgG, 1:5000 dilution was used) in PBS-MLK for 1.5 hours at room temperature with agitation. 6. Wash the nitrocellulose with water twice. 7. Wash the nitrocellulose in PBS-0.05% Tween 20 for 3-15 minutes. 8. Rinse the nitrocellulose in water for 30-60min. 9. Use detection method of choice (enhanced chemiluminescence was used).
Storage and StabilityMay be stored at 4°C for short-term only. For long-term storage and to avoid repeated freezing and thawing, aliquot and add glycerol (40-50%). Freeze at -20°C. Aliquots are stable for at least 12 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
Clone TypePolyclonal
IsotypeIgG
HostRabbit
SourceHuman
ConcentrationNot determined
FormSupplied as a liquid in 0.2M Tris-glycine, pH 7.4, 0.15M NaCl, 0.05% sodium azide.
PurityPurified by immunoaffinity chromatography.
ImmunogenSynthetic phosphopeptide, residues 1243-1255, corresponding to a major auto-phosphorylation (Tyr1248) site of human Erb B2/HER-2 (C-AENPEpYLGLDVPV).
SpecificityRecognizes the activated form of human Erb B2/HER-2 oncoprotein at 185 kD, phosphorylated at Tyr1248. Species Crossreactivity: human. Predicted to crossreact with rat based on conservation of immunogenic sequence.
Important NoteThis product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.


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