Product datasheet
Anti-Notch1 antibody ab65297
2 Abreviews 1 References 6 Images
Overview
Product name
Anti-Notch1 antibody
Description
Rabbit polyclonal to Notch1
Tested applications
ICC/IF, IHC-Fr, IP, IHC-P, WB, Flow Cyt
Species reactivity
Reacts with: Mouse, Human
Immunogen
Synthetic peptide conjugated to KLH derived from within residues 2500 to the C-terminus of
Human Notch1.Read Abcam's proprietary immunogen policy(Peptide available as ab68580.)
Positive control
This antibody gave a positive signal in the following whole cell lysates: HeLa, HepG2, HEK293,
SK N SH and Jurkat. This antibody gave a positive result when used in the following
formaldehyde fixed cell lines: HepG2.
Properties
Form
Liquid
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or 80°C. Avoid freeze / thaw cycle.
Storage buffer
Preservative: 0.02% Sodium Azide
Constituents: 1% BSA, PBS, pH 7.4
Purity
Immunogen affinity purified
Clonality
Polyclonal
Isotype
IgG
Applications
Our Abpromise guarantee covers the use of ab65297 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application
Abreviews
Notes
ICC/IF
Use a concentration of 5 µg/ml.
IHC-Fr
1/100.
IP
Use a concentration of 5 µg/ml.
IHC-P
Use a concentration of 1 µg/ml.
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Application
WB
Abreviews
Notes
Use a concentration of 1 µg/ml. Detects a band of approximately 120 kDa
(predicted molecular weight: 272 kDa).
Flow Cyt
Use at an assay dependent concentration. PubMed: 24136221ab171870-Rabbit
polyclonal IgG, is suitable for use as an isotype control with this antibody.
Target
Function
Functions as a receptor for membrane-bound ligands Jagged1, Jagged2 and Delta1 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. May be important for normal lymphocyte function. In altered form, may
contribute to transformation or progression in some T-cell neoplasms. Involved in the maturation
of both CD4+ and CD8+ cells in the thymus. May be important for follicular differentiation and
possibly cell fate selection within the follicle. During cerebellar development, may function as a
receptor for neuronal DNER and may be involved in the differentiation of Bergmann glia.
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.
Involvement in disease
Defects in NOTCH1 are a cause of bicuspid aortic valve (BAV) [MIM:109730]. A common defect
in the aortic valve in which two rather than three leaflets are present. It is often associated with
aortic valve calcification and insufficiency. In extreme cases, the blood flow may be so restricted
that the left ventricle fails to grow, resulting in hypoplastic left heart syndrome.
Sequence similarities
Belongs to the NOTCH family.
Contains 5 ANK repeats.
Contains 36 EGF-like domains.
Contains 3 LNR (Lin/Notch) repeats.
Post-translational
modifications
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. Cleavage results in a C-terminal fragment N(TM) and a
N-terminal fragment N(EC). Following ligand binding, it is cleaved 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 gammasecretase to release a notch-derived peptide containing the intracellular domain (NICD) from the
membrane.
Phosphorylated.
O-glycosylated on the EGF-like domains. Contains both O-linked fucose and O-linked glucose.
Ubiquitinated; undergoes 'Lys-29'-linked polyubiquitination catalyzed by ITCH.
Cellular localization
Cell membrane and Nucleus. Following proteolytical processing NICD is translocated to the
nucleus.
Anti-Notch1 antibody images
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Notch1 was immunoprecipitated using 0.5mg
Hek293 whole cell extract, 5µg of Rabbit
polyclonal to Notch1 and 50µl of protein G
magnetic beads (+). No antibody was added
to the control (-).
The antibody was incubated under agitation
with Protein G beads for 10min, Hek293
whole cell extract lysate diluted in RIPA buffer
Immunoprecipitation - Anti-Notch1 antibody
(ab65297)
was added to each sample and incubated for
a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS
loading buffer and incubated for 10min at
70°C; 10µl of each sample was separated on
a SDS PAGE gel, transferred to a
nitrocellulose membrane, blocked with 5%
BSA and probed with ab65297.
Secondary: Mouse monoclonal [SB62a]
Secondary Antibody to Rabbit IgG light chain
(HRP) (ab99697).
Band: 120kDa; Notch1
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All lanes : Anti-Notch1 antibody (ab65297) at
1 µg/ml
Lane 1 : HeLa (Human epithelial carcinoma
cell line) Whole Cell Lysate
Lane 2 : HepG2 (Human hepatocellular liver
carcinoma cell line) Whole Cell Lysate
Lane 3 : HEK293 (Human embryonic kidney
cell line) Whole Cell Lysate
Lane 4 : SK N SH (Human neuroblastoma)
Whole Cell Lysate
Western blot - Notch1 antibody (ab65297)
Lane 5 : Jurkat (Human T cell lymphoblastlike cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Secondary
Goat polyclonal to Rabbit IgG - H&L - PreAdsorbed (HRP) at 1/3000 dilution
Performed under reducing conditions.
Predicted band size : 272 kDa
Observed band size : 120 kDa
Additional bands at : 160 kDa,50 kDa. We
are unsure as to the identity of these extra
bands.
Exposure time : 3 minutesThe band
observed at 120 kDa could potentially be a
truncated form of Notch1, following cleavage
by furin-like protease. This band is also
observed with other commercially available
antibodies to Notch1.
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IHC image of Notch1 staining in Human
Hippocampus FFPE section, performed on a
BondTM system using the standard protocol
F. The section was pre-treated using heat
mediated antigen retrieval with sodium citrate
buffer (pH6, epitope retrieval solution 1) for 20
mins. The section was then incubated with
ab65297, 1µg/ml, for 15 mins at room
Immunohistochemistry (Formalin/PFA-fixed
temperature and detected using an HRP
paraffin-embedded sections) - Notch1 antibody
conjugated compact polymer system. DAB
(ab65297)
was used as the chromogen. The section was
then counterstained with haematoxylin and
mounted with DPX
ICC/IF image of ab65297 stained HepG2
cells. The cells were 4% formaldehyde fixed
(10 min) and then incubated in 1%BSA / 10%
normal goat serum / 0.3M glycine in 0.1%
PBS-Tween for 1h to permeabilise the cells
and block non-specific protein-protein
interactions. The cells were then incubated
with the antibody ab65297 at 5µg/ml
overnight at +4°C. The secondary antibody
Immunocytochemistry/ Immunofluorescence -
(green) was DyLight® 488 goat anti- rabbit
Anti-Notch1 antibody (ab65297)
(ab96899) IgG (H+L) used at a 1/250 dilution
for 1h. Alexa Fluor® 594 WGA was used to
label plasma membranes (red) at a 1/200
dilution for 1h. DAPI was used to stain the cell
nuclei (blue) at a concentration of 1.43µM.
ab65297 staining Notch1 (green) in
Mouse embryo tissue sections by
Immunohistochemistry (IHC-P paraformaldehyde-fixed, paraffin-embedded
sections). Tissue was fixed with formaldehyde
and blocked with 5% serum for 1 hour at room
temperature; antigen retrieval was by heat
Immunohistochemistry (Formalin/PFA-fixed
mediation in a 0.5% citrate buffer. Samples
paraffin-embedded sections) - Anti-Notch1
were incubated with primary antibody (1/50
antibody (ab65297)
in 5% Goat serum) for 2 hours. An Alexa
This image is courtesy of an anonymous Abreview
Fluor® 488-conjugated Goat anti-rabbit IgG
polyclonal (1/300) was used as the secondary
antibody. Red - Myh6/7, Blue - nuclei (DAPI).
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ab65297 staining Notch1 (green) in
Mouse embryo tissue sections by
Immunohistochemistry (IHC-Fr - frozen
sections). Tissue was fixed with
paraformaldehyde, permeabilized with 1%
Triton X-100 and blocked with 5% serum for 2
hours at room temperature. Samples were
Immunohistochemistry (Frozen sections) - AntiNotch1 antibody (ab65297)
This image is courtesy of an anonymous Abreview
incubated with primary antibody (1/100 in 5%
Goat serum) for 2 hours. An Alexa Fluor®
488-conjugated Goat anti-rabbit IgG
polyclonal (1/400) was used as the secondary
antibody. Red - Myh6/7, Blue - nuclei (DAPI).
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