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Notch 1 Recombinant Rabbit Monoclonal Antibody [SJ205]

Usd: 205

Specification

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Catalog# ET1606-55

Notch 1 Recombinant Rabbit Monoclonal Antibody [SJ205]

Application

  • WB

  • IF-Cell

  • IF-Tissue

  • IHC-P

  • IP

  • FC

Reactivity

  • Human

  • Mouse

  • Rat

BSA and Azide free
Conjugation

  • unconjugated

This product has been cited in peer reviewed publications, see list HERE

Overview

Product Name

Notch 1 Recombinant Rabbit Monoclonal Antibody [SJ205]

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic peptide within Human Notch 1 aa 2,481-2,530 / 2,555.

Species Reactivity

Human, Mouse, Rat

Validated Applications

WB, IF-Cell, IF-Tissue, IHC-P, IP, FC

Molecular Weight

Predicted band size: 273 kDa

Positive Control

C6 cell lysate, rat brain tissue lysate, HeLa cell lysate, THP-1 cell lysate, A549 cell lysate, HEK-293 cell lysate, Hela, HepG2, NIH/3T3, human pancreas tissue, mouse lung tissue, mouse pancreas tissue.

Conjugation

unconjugated

Clone Number

SJ205

RRID

AB_3069748

Product Features

Form

Liquid

Concentration

Storage Instructions

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). Store at -20℃ long term.

Storage Buffer

1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

  • WB

  • 1:1,000-1:5,000

  • IF-Cell

  • 1:50-1:200

  • IF-Tissue

  • 1:50-1:200

  • IHC-P

  • 1:50-1:200

  • FC

  • 1:50-1:100

  • IP

  • Use at an assay dependent concentration.

Target

Function

The LIN-12/notch family of transmembrane receptors is believed to play a central role in development by regulating cell fate decisions. To date, four notch homologs have been identified in mammals and have been designated Notch 1, Notch 2, Notch 3 and Notch 4. The notch genes are expressed in a variety of tissues in both the embryonic and adult organism, suggesting that the genes are involved in multiple signaling pathways. The notch proteins have been found to be overexpressed or rearranged in human tumors. Ligands for notch include Jagged1, Jagged2 and Delta. Jagged can activate notch and prevent myoblast differentiation by inhibiting the expression of muscle regulatory and structural genes. Jagged2 is thought to be involved in the development of various tissues whose development is dependent upon epithelial-mesenchymal interactions. Normal Delta expression is restricted to the adrenal gland and placenta. Delta expression has also been found in neuroendocrine tumors such as neuroblastomas and pheochromocytomas.

Background References

1. Li W et al. Genome-wide analyses identify KLF4 as an important negative regulator in T-cell acute lymphoblastic leukemia through directly inhibiting T-cell associated genes. Mol Cancer 14:26 (2015).

2. Nakajima K et al. Galectin-3 inhibits osteoblast differentiation through notch signaling. Neoplasia 16:939-49 (2014).

Sequence Similarity

Belongs to the NOTCH family.

Tissue Specificity

In fetal tissues most abundant in spleen, brain stem and lung. Also present in most adult tissues where it is found mainly in lymphoid tissues.

Post-translational Modification

Synthesized in the endoplasmic reticulum as an inactive form which is proteolytically cleaved by a furin-like convertase in the trans-Golgi network before it reaches the plasma membrane to yield an active, ligand-accessible form (By similarity). Cleavage results in a C-terminal fragment N(TM) and a N-terminal fragment N(EC). Following ligand binding, it is cleaved by ADAM17 to yield a membrane-associated intermediate fragment called notch extracellular truncation (NEXT). Following endocytosis, this fragment is then cleaved by one of the catalytic subunits of gamma-secretase (PSEN1 or PSEN2), to release a Notch-derived peptide containing the intracellular domain (NICD) from the membrane.; Phosphorylated.; O-glycosylated on the EGF-like domains. O-glucosylated at Ser-435 by KDELC1 and KDELC2. Contains both O-linked fucose and O-linked glucose in the EGF-like domains 11, 12 and 13, which are interacting with the residues on DLL4 (By similarity). O-linked glycosylation by GALNT11 is involved in determination of left/right symmetry: glycosylation promotes activation of NOTCH1, possibly by promoting cleavage by ADAM17, modulating the balance between motile and immotile (sensory) cilia at the left-right organiser (LRO). MFNG-, RFNG- and LFNG-mediated modification of O-fucose residues at specific EGF-like domains results in inhibition of its activation by JAG1 and enhancement of its activation by DLL1 via an increased binding to DLL1 (By similarity).; Ubiquitinated. Undergoes 'Lys-29'-linked polyubiquitination by ITCH; promotes the lysosomal degradation of non-activated internalized NOTCH1. Monoubiquitination at Lys-1759 is required for activation by gamma-secretase cleavage, it promotes interaction with AAK1, which stabilizes it. Deubiquitination by EIF3F is necessary for nuclear import of activated Notch.; Hydroxylated at Asn-1955 by HIF1AN. Hydroxylated at Asn-2022 by HIF1AN (By similarity). Hydroxylation reduces affinity for HI1AN and may thus indirectly modulate negative regulation of NICD (By similarity).

Subcellular Location

Cell membrane, Late endosome membrane; Nucleus (intracellular domain).

UNIPROT

SwissProt: P46531 Human

SwissProt: Q01705 Mouse

SwissProt: Q07008 Rat

Synonyms

9930111A19Rik antibody

AOS5 antibody

AOVD1 antibody

hN1 antibody

Lin-12 antibody

LIN12 antibody

Mis6 antibody

Motch A antibody

mT14 antibody

Neurogenic locus notch homolog protein 1 antibody

Expand

Images

  • Western blot analysis of Notch 1 on different lysates with Rabbit anti-Notch 1 antibody (<a href="/products/ET1606-55" style="font-weight: bold;text-decoration: underline;">ET1606-55</a>) at 1/5,000 dilution.<br /><br />Lane 1: C6 cell lysate (20 µg/Lane)<br />Lane 2: Rat brain tissue lysate (40 µg/Lane)<br /><br />Predicted band size: 273 kDa<br />Observed band size: 125/273 kDa<br /><br />Exposure time: 1 minute; ECL: K1801;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1606-55" style="font-weight: bold;text-decoration: underline;">ET1606-55</a>) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of Notch 1 on different lysates with Rabbit anti-Notch 1 antibody (ET1606-55) at 1/5,000 dilution.

    Lane 1: C6 cell lysate (20 µg/Lane)
    Lane 2: Rat brain tissue lysate (40 µg/Lane)

    Predicted band size: 273 kDa
    Observed band size: 125/273 kDa

    Exposure time: 1 minute; ECL: K1801;

    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1606-55) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • Western blot analysis of Notch 1 on different lysates with Rabbit anti-Notch 1 antibody (<a href="/products/ET1606-55" style="font-weight: bold;text-decoration: underline;">ET1606-55</a>) at 1/1,000 dilution.<br /><br />Lane 1: HeLa cell lysate<br />Lane 2: THP-1 cell lysate<br />Lane 3: A549 cell lysate<br />Lane 4: HEK-293 cell lysate<br /><br />Lysates/proteins at 30 µg/Lane.<br /><br />Predicted band size: 273 kDa<br />Observed band size: 125/273 kDa<br /><br />Exposure time: 59 seconds; ECL: K1801;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1606-55" style="font-weight: bold;text-decoration: underline;">ET1606-55</a>) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of Notch 1 on different lysates with Rabbit anti-Notch 1 antibody (ET1606-55) at 1/1,000 dilution.

    Lane 1: HeLa cell lysate
    Lane 2: THP-1 cell lysate
    Lane 3: A549 cell lysate
    Lane 4: HEK-293 cell lysate

    Lysates/proteins at 30 µg/Lane.

    Predicted band size: 273 kDa
    Observed band size: 125/273 kDa

    Exposure time: 59 seconds; ECL: K1801;

    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1606-55) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • <span style="font-weight: bold;">☑ Knockdown (KD)</span><br /><br />Western blot analysis of Notch 1 on different lysates with Rabbit anti-Notch 1 antibody (<a href="/products/ET1606-55" style="font-weight: bold;text-decoration: underline;">ET1606-55</a>) at 1/1,000 dilution.<br /><br />Lane 1: Hela-si NT cell lysate<br />Lane 2: Hela-si Notch 1 cell lysate<br /><br />Lysates/proteins at 20 µg/Lane.<br /><br />Predicted band size: 273 kDa<br />Observed band size: 125 kDa<br /><br />Exposure time: 6 minutes;<br /><br />4-20% SDS-PAGE gel.<br /><br /><a href="/products/ET1606-55" style="font-weight: bold;text-decoration: underline;">ET1606-55</a> was shown to specifically react with Notch 1 in Hela-si NT cells. Weakened band was observed when Hela-si Notch 1 sample was tested. Hela-si NT and Hela-si Notch 1 samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1606-55" style="font-weight: bold;text-decoration: underline;">ET1606-55</a>, 1/1,000) and Loading control antibody (Rabbit anti-GAPDH, <a href="/products/ET1601-4" style="font-weight: bold;text-decoration: underline;">ET1601-4</a>, 1/10,000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-rabbit IgG-HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1:100,000 dilution was used for 1 hour at room temperature.

    ☑ Knockdown (KD)

    Western blot analysis of Notch 1 on different lysates with Rabbit anti-Notch 1 antibody (ET1606-55) at 1/1,000 dilution.

    Lane 1: Hela-si NT cell lysate
    Lane 2: Hela-si Notch 1 cell lysate

    Lysates/proteins at 20 µg/Lane.

    Predicted band size: 273 kDa
    Observed band size: 125 kDa

    Exposure time: 6 minutes;

    4-20% SDS-PAGE gel.

    ET1606-55 was shown to specifically react with Notch 1 in Hela-si NT cells. Weakened band was observed when Hela-si Notch 1 sample was tested. Hela-si NT and Hela-si Notch 1 samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (ET1606-55, 1/1,000) and Loading control antibody (Rabbit anti-GAPDH, ET1601-4, 1/10,000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-rabbit IgG-HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature.

  • ICC staining of Notch 1 in Hela cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (<a href="/products/ET1606-55" style="font-weight: bold;text-decoration: underline;">ET1606-55</a>, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor&reg;488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

    ICC staining of Notch 1 in Hela cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1606-55, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

  • ICC staining of Notch 1 in HepG2 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (<a href="/products/ET1606-55" style="font-weight: bold;text-decoration: underline;">ET1606-55</a>, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor&reg;488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

    ICC staining of Notch 1 in HepG2 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1606-55, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

  • ICC staining of Notch 1 in NIH/3T3 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (<a href="/products/ET1606-55" style="font-weight: bold;text-decoration: underline;">ET1606-55</a>, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor&reg;488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

    ICC staining of Notch 1 in NIH/3T3 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1606-55, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

  • Immunohistochemical analysis of paraffin-embedded human pancreas tissue using anti-Notch 1 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1606-55" style="font-weight: bold;text-decoration: underline;">ET1606-55</a>, 1/200) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded human pancreas tissue using anti-Notch 1 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1606-55, 1/200) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded mouse lung tissue using anti-Notch 1 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1606-55" style="font-weight: bold;text-decoration: underline;">ET1606-55</a>, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded mouse lung tissue using anti-Notch 1 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1606-55, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded mouse pancreas tissue using anti-Notch 1 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1606-55" style="font-weight: bold;text-decoration: underline;">ET1606-55</a>, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded mouse pancreas tissue using anti-Notch 1 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1606-55, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunofluorescence analysis of paraffin-embedded human liver cancer tissue labeling Notch 1 with Rabbit anti-Notch 1 antibody (<a href="/products/ET1606-55" style="font-weight: bold;text-decoration: underline;">ET1606-55</a>) at 1/50 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (<a href="/products/ET1606-55" style="font-weight: bold;text-decoration: underline;">ET1606-55</a>, green) at 1/50 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).

    Immunofluorescence analysis of paraffin-embedded human liver cancer tissue labeling Notch 1 with Rabbit anti-Notch 1 antibody (ET1606-55) at 1/50 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (ET1606-55, green) at 1/50 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).

  • Flow cytometric analysis of Notch 1 was done on Hela cells. The cells were fixed, permeabilized and stained with the primary antibody (<a href="/products/ET1606-55" style="font-weight: bold;text-decoration: underline;">ET1606-55</a>, 1/50) (blue). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Rabbit IgG Secondary antibody at 1/1000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; red).

    Flow cytometric analysis of Notch 1 was done on Hela cells. The cells were fixed, permeabilized and stained with the primary antibody (ET1606-55, 1/50) (blue). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Rabbit IgG Secondary antibody at 1/1000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; red).

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Citation

Products with the same target and pathway

metafield image

Notch 1 Recombinant Rabbit Monoclonal Antibody [SJ205] - BSA and Azide free

Application: WB,IF-Cell,IF-Tissue,IHC-P,IP,FC

Reactivity: Human,Mouse,Rat

Conjugate: unconjugated