NOTCH2 Antikörper (N-Term)

Produktnummer ABIN104899

Produktdetails anti-NOTCH2 Antikörper

Target: NOTCH2 (Notch 2 (NOTCH2))

Bindungsspezifität: N-Term

Reaktivität: Human

Wirt: Kaninchen

Klonalität: Polyklonal

Konjugat: Dieser NOTCH2 Antikörper ist unkonjugiert

Applikation: Western Blotting (WB), ELISA

Spezifität: This antiserum is directed against human NOTCH 2. Based on sequences, we expect this antibody to react as well with rat and mouse NOTCH 2. Only one amino acid difference (conservative) occurs in mouse and rat. The immunogen epitope is only exposed after gamma secretase cleavage and is not accessible in the uncleaved form.

Produktmerkmale: Anti Notch 2 Antibody recognizes Notch 2 that is synthesized in the endoplasmic reticulum as an inactive form which is proteolytically cleaved by a furin-like convertase (S1 cleavage) in the trans-golgi network before it reaches the plasma membrane to yield an active, ligand-accessible form. Cleavage results in a C-terminal fragment N(TM) and a N-terminal fragment N(EC). Following ligand binding, it is cleaved (S2 cleavage) by TNF-alpha converting enzyme (TACE) to yield a membrane-associated intermediate fragment called Notch extracellular truncation (NEXT). This fragment is then cleaved by presenilin-dependent gamma-secretase (S3 cleavage) to release the intracellular domain (NICD) from the membrane.

Sterilität: Sterile filtered

Immunogen: This whole rabbit serum was prepared by repeated immunizations with a synthetic peptide corresponding to amino acid residues of human Notch 2 located near the N-terminal sequence of the cleaved N intracellular domain (NICD).
Immunogen Type: Peptide

Produktspezifische Information:

Wofür kann der NOTCH2-Antikörper ABIN104899 verwendet werden? Dieser polyklonale, nicht konjugierte Kaninchen-anti-NOTCH2-Antikörper weist humanes NOTCH2 im ELISA und Western Blotting zuverlässig nach.

Welche Validierungsdaten liegen für diesen NOTCH2-Antikörper vor? Für den Western-Blotting-Test liegt derzeit eine unabhängige Validierung vor, die an der Universität Ulm, Deutschland, durchgeführt wurde. Aufgrund der Sequenz kann davon ausgegangen werden, dass der NOTCH2-Antikörper auch bei Ratten und Mäusen funktioniert. Der Antikörper wurde bisher in drei Veröffentlichungen verwendet, die unten aufgeführt sind und bei PubMed nachgelesen werden können. Es sind 2 Bilder verfügbar, die die Leistung des NOTCH2-Antikörpers im Western Blot zeigen. Verwenden Sie unseren NOTCH2-Antikörper, um humanes NOTCH 2 zuverlässig nachzuweisen.

Was ist die Funktion von NOTCH2? NOTCH2 fungiert als Rezeptor für die membrangebundenen Liganden Jagged-1 (JAG1), Jagged-2 (JAG2) und Delta-1 (DLL1) und steuert so die Bestimmung des Zellschicksals. Nach Ligandenaktivierung durch die freigesetzte intrazelluläre Notch-Domäne (NICD) bildet es einen Transkriptionsaktivatorkomplex mit RBPJ/RBPSUH und aktiviert Gene des Enhancer of Split Locus. Beeinflusst die Umsetzung von Differenzierungs-, Proliferations- und Apoptoseprogrammen (By similarity). NOTCH2 scheint am Knochenumbau und an der Homöostase beteiligt zu sein. In Zusammenarbeit mit RELA/p65 verstärkt es die Aktivität des NFATc1-Promotors und reguliert positiv die RANKL-induzierte Osteoklastendifferenzierung. (UniProt)

Anwendungsinformationen

Applikationshinweise: A strong response was detected by ELISA against the immunizing peptide.This product was assayed against the peptide immunogen in a standard capture ELISA using Peroxidase conjugated anti-Rabbit IgG [H&L] Goat) and ABTS (2,2’-azino-bis-[3-ethylbenthiazoline-6-sulfonic acid]) as a substrate for 30 minutes at room temperature. A working dilution of 1:30,000 to 1:90,000 is suggested for this product. This product was assayed against the peptide immunogen in a standard capture ELISA using Peroxidase conjugated anti-Rabbit IgG [H&L] (Goat) ( and ABTS (2,2’-azino-bis-[3-ethylbenthiazoline-6-sulfonic acid]) ( as a substrate for 30 minutes at room temperature. A working dilution of 1:30,000 to 1:90,000 is suggested for this product.

Kommentare:

Gene Name: NOTCH2

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (WB)

Validierung #103703 (Western Blotting)

by: AG Pancreatic Development and Stem cell differentiation, Universitätsklinikum Ulm

No.: #103703

Datum: 16.06.2019

Antigen: NOTCH2

Validierte Anwendung: Western Blotting

Positivkontrolle:

pancreatic endoderm cells (day 10)

pancreatic progenitor cells (day 14)

Negativkontrolle:

human pluripotent stem cells (day 0)

defnite endoderm cells (day 4)

Bewertung:

ABIN1043688 reveals two protein bands of the expected size and some weaker extraneous bands in cell lysates of human pluripotent stem cells, definite endoderm cells, pancreatic endoderm cells, and pancreatic progenitor cells.

Validierungsbilder

Western blot with ABIN1043688 on human pluripotent stem cells (1 and 2), definite endoderm cells (3 and 4), pancreatic endoderm cells (5 and 6), and pancreatic progenitors (7 and 8). ABIN1043688 was used at a 1:1000 dilution. Vinculin served as loading control.

Protokoll

Primärantikörper: ABIN1043688

Sekundärantikörper: donkey anti-rabbit HRP-conjugated antibody (GE Healthcare, NA9310V)

Full Protocol:

• Grow HUES8 in mTeSR1 (STEMCELL Technologies, 85850) at 37°C and 5% O₂, 5% CO₂ in 2ml in a 6-well plate to 90% confluency.
• Harvest cells using TrypLE (ThermoFisher Scientific, 12604013) following the manufacturer´s instructions.
• Lyse 2x10⁶ cells in 30µl per well cold RIPA buffer (50mM Tris-HCl pH 8.0, 150mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% TritonX 100 in ddH₂O) supplemented with 1mM PMSF and 1x protease inhibitor (Roche, 11836170001) for 30min on ice with 3x vortexing in 1.5ml microcentrifuge tubes.
• Centrifuge tubes at 10000xg for 8min at 4°C.
• Transfer supernatant to a new 1.5ml microcentrifuge tube and store at -80°C.
• Determine total protein content of the lysates using a Bradford assay (Bio-Rad, 500-0006).
• Denature 50µg of total protein for 5min at 95°C in 30µl 1x Laemmli SDS sample buffer and subsequently separate them on a denaturing 7.5% polyacrylamide gel (7.5% Acrylamide, 0.375M Tris-HCl pH8.8, 0.1% SDS, 0.1% APS, 0.1% TEMED) for about 120min at 120V.
• Transfer proteins onto a PVDF membrane (Sigma Aldrich, IPVH00010) with transfer buffer (5.27g Tris, 2.93 g glycerine, 200 ml methanol, fill to 1l ddH₂O) in a semidry western blotting system for 80min at 80mA/gel.
• Check transfer of the separated proteins by Ponceau S staining.
• Rinse membrane with water.
• Wash membrane for 5min with TBST (TBS, 0.02% Tween20).
• Block the membrane with 20ml blocking buffer (TBST, 5% milk) on a shaker for 1h at RT.
• Rinse membrane 3x with TBST.
• Wash membrane on a shaker 3x for 5min with TBST.
• Shrink-wrap and incubate membrane with primary
• • rabbit anti-NOTCH2 antibody (antibodies-online, ABIN1043688) diluted 1:1000 respectively in blocking buffer ON at 4°C or
  • mouse anti-vinculin antibody (SigmaAldrich, V9264) diluted 1:1000 in blocking buffer at RT for 1h.
• Wash membrane 3x for 5min with TBST.
• Incubate membrane with secondary
• • donkey anti-rabbit HRP-conjugated antibody (GE Healthcare, NA9310V) diluted 1:5000 in TBST containing 1% milk for 1h at RT or
  • donkey anti-mouse HRP-conjugated antibody (GE Healthcare, NA9340V) diluted 1:5000 in TBST containing 1% milk for 1h at RT.
• Wash membrane 3x for 5min with TBST.
• Reveal protein bands using ECL solution (ThermoScientific, 34076) with an exposure time of 40min for the ABIN1043688 and 2min for the vinculin loading control.

Anmerkungen:

• ABIN1043688 revealed some strong unspecific bands. Nevertheless, a band over 250kDa appears to be highly specific, as it is only detected at day 10 (lanes 5 and 6) and day 14 (lanes 7 and 8).

• Manual exposure had to be performed due to a defect of the fusion detector in our institute.

• ABIN1043688 was also tested in IHC on 4-5µm FFPE sections of primary human kidney cancer tissue. Epitope retrieval was carried out using Tris-EDTA buffer at pH9.0 (Zytomed, ZUC029-500), EDTA at pH8.0 (Leica, RE7116), or citrate buffer at pH6.1 (Agilent, S169984-2) for 20min in a decloaking chamber.

• Interpretation of the results was difficult, as the method of antigen retrieval strongly effects the staining pattern. Antigen retrieval using the citrate buffer at pH6 might be ok, but knockout or knockdown studies are necessary to clearly conclusively show specificity.

Handhabung

Format: Liquid

Konzentration: 70 mg/mL

Buffer: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2

Konservierungsmittel: Sodium azide

Vorsichtsmaßnahmen: This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Lagerung: 4 °C/-20 °C

Informationen zur Lagerung: Store vial at 4 °C prior to restoration. For extended storage aliquot contents and freeze at -20 °C or below. 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. Expiration date is one (1) year from date of opening.

Haltbarkeit: 12 months

Referenzen für anti-NOTCH2 Antikörper (ABIN104899)

Iervolino, Prosperi, De La Motte, Petrillo, Spagnuolo, DAcierno, Siccardi, Perna, Christensen, Frische, Capasso, Trepiccione: "Potassium depletion induces cellular conversion in the outer medullary collecting duct altering Notch signaling pathway." in: Scientific reports, Vol. 10, Issue 1, pp. 5708, (2020) (PubMed).

Gridley: "Notch signaling and inherited disease syndromes." in: Human molecular genetics, Vol. 12 Spec No 1, pp. R9-13, (2003) (PubMed).

Bertrand, Eckfeldt, Lysholm, LeBien: "Notch-1 and Notch-2 exhibit unique patterns of expression in human B-lineage cells." in: Leukemia, Vol. 14, Issue 12, pp. 2095-102, (2001) (PubMed).

Details zu NOTCH2

Target: NOTCH2 (Notch 2 (NOTCH2))

Andere Bezeichnung: NOTCH 2 (NOTCH2 Produkte)

Synonyme: AGS2 antikoerper, HJCYS antikoerper, hN2 antikoerper, AI853703 antikoerper, N2 antikoerper, notch6 antikoerper, sb:cb884 antikoerper, neurogenic locus notch homolog protein 2 antikoerper, notch 2 antikoerper, notch 3 antikoerper, LOC581186 antikoerper, NOTCH2 antikoerper, notch3 antikoerper, Notch2 antikoerper, notch2 antikoerper

Hintergrund: Anti Notch 2 Antibody recognizes Notch 2 that is synthesized in the endoplasmic reticulum as an inactive form which is proteolytically cleaved by a furin-like convertase (S1 cleavage) in the trans-golgi network before it reaches the plasma membrane to yield an active, ligand-accessible form. Cleavage results in a C-terminal fragment N(TM) and a N-terminal fragment N(EC). Following ligand binding, it is cleaved (S2 cleavage) by TNF-alpha converting enzyme (TACE) to yield a membrane-associated intermediate fragment called Notch extracellular truncation (NEXT). This fragment is then cleaved by presenilin-dependent gamma-secretase (S3 cleavage) to release the intracellular domain (NICD) from the membrane.
Synonyms: AGS2 antibody, hN2 antibody, Notch homolog 2 antibody, Notch2 antibody

Gen-ID: 4853, 24041035

UniProt: Q04721

Pathways: Notch Signalweg, Stem Cell Maintenance