NOTCH2 antibody (N-Term)

Catalog No. ABIN104899

Product Details anti-NOTCH2 Antibody

Target: NOTCH2 (Notch 2 (NOTCH2))

Binding Specificity: N-Term

Reactivity: Human

Host: Rabbit

Clonality: Polyclonal

Conjugate: This NOTCH2 antibody is un-conjugated

Application: Western Blotting (WB), ELISA

Specificity: 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.

Characteristics: 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.

Sterility: 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

Product Specific Information:

What can the NOTCH2 antibody ABIN104899 be used for? This polyclonal, unconjugated Rabbit anti-NOTCH2 antibody reliably detects human NOTCH2 by ELISA and Western blotting.

What validation data is available for this NOTCH2 antibody? For western blotting, independent validation is currently available and was performed at the University of Ulm, Germany. Based on the sequence, it can be assumed that the NOTCH2 antibody also works for rat and mouse species. The antibody has been used in three publications to date, which are given below and can be read at PubMed. There are 2 images available that demonstrate performance of the NOTCH2 antibody in Western blot. Use our NOTCH2 antibody to reliably detect human NOTCH 2.

What is the function of NOTCH2? NOTCH2 functions as a receptor for membrane-bound ligands Jagged-1 (JAG1), Jagged-2 (JAG2) and Delta-1 (DLL1) to regulate cell-fate determination. Upon ligand activation through the released notch intracellular domain (NICD) it forms a transcriptional activator complex with RBPJ/RBPSUH and activates genes of the enhancer of split locus. Affects the implementation of differentiation, proliferation and apoptotic programs (By similarity). NOTCH2 seems to be involved in bone remodeling and homeostasis. In collaboration with RELA/p65 enhances NFATc1 promoter activity and positively regulates RANKL-induced osteoclast differentiation. (UniProt)

Application Details

Application Notes: 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.

Comment:

Gene Name: NOTCH2

Restrictions: For Research Use only

Independent Validation (WB)

Validation #103703 (Western Blotting)

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

No.: #103703

Date: 06/16/2019

Antigen: NOTCH2

Method validated: Western Blotting

Positive Control:

pancreatic endoderm cells (day 10)

pancreatic progenitor cells (day 14)

Negative Control:

human pluripotent stem cells (day 0)

defnite endoderm cells (day 4)

Notes:

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.

Validation Images

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.

Full Methods

Primary Antibody: ABIN1043688

Secondary Antibody: 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.

Experimental Notes:

• 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.

Handling

Format: Liquid

Concentration: 70 mg/mL

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

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: 4 °C/-20 °C

Storage Comment: 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.

Expiry Date: 12 months

References for anti-NOTCH2 antibody (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).

Target Details for NOTCH2

Target: NOTCH2 (Notch 2 (NOTCH2))

Alternative Name: NOTCH 2 (NOTCH2 Products)

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

Background: 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

Gene ID: 4853, 24041035

UniProt: Q04721

Pathways: Notch Signaling, Stem Cell Maintenance