Overview

  • Product name
    Anti-activated Notch1 antibody
    See all activated Notch1 primary antibodies
  • Description
    Rabbit polyclonal to activated Notch1
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IHC-P, IHC-Fr, ICC/IF, WB, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Human
    Predicted to work with: Rat
  • Immunogen

    Synthetic peptide corresponding to activated Notch1 aa 1755-1767 (intracellular). The epitope is only exposed after gamma secretase cleavage and is not accessible in the uncleaved form.
    Sequence:

    VLLSRKRRRQHGQC


    (Peptide available as ab730)

  • Positive control
    • WB: Lysates from myc-tagged transiently transfected mouse Notch constructs in HEK-293 cells. IHC-Fr: Mouse dermis. IHC-P: Human ovarian carcinoma tissue. ICC/IF: NIH/3T3 cells.
  • General notes

    Notch 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.

Properties

Applications

Our Abpromise guarantee covers the use of ab8925 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
IHC-P 1/200.
IHC-Fr Use at an assay dependent concentration. PubMed: 18371421
ICC/IF Use at an assay dependent concentration.
WB 1/500. Detects a band of approximately 80 kDa.Can be blocked with Human activated Notch1 peptide (ab730). 80kDa band is a cleavage product of Notch 1. There is also a contaminating band present at 100 kDa. The identity of this band is unknown.
Flow Cyt Use at an assay dependent concentration. PubMed: 19171875

ab171870 - 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. Represses neuronal and myogenic differentiation. May enhance HIF1A function by sequestering HIF1AN away from HIF1A.
  • 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 aortic valve disease 1 (AOVD1) [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). Following endocytosis, this fragment is then cleaved by presenilin dependent gamma-secretase 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. 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.
  • Cellular localization
    Cell membrane and Nucleus. Following proteolytical processing NICD is translocated to the nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • hN1 antibody
    • Neurogenic locus Notch homolog protein 1 antibody
    • NICD antibody
    • NOTC1_HUMAN antibody
    • Notch 1 antibody
    • Notch 1 intracellular domain antibody
    • Notch homolog 1 translocation associated (Drosophila) antibody
    • notch1 antibody
    • TAN1 antibody
    • Translocation-associated notch protein TAN-1 antibody
    see all

Images

  • The Notch signaling pathway is activated in the crypts.

    (Panel B) Immunofluorescence staining for NICD (red, ab8925), with β-catenin in green and DAPI in blue.

    For immunofluorescent staining of mouse small intestine cryosections, we used a blocking solution of 1% BSA and 0.1% Triton X-100 in PBS.

  • TGFβ/Smad transcriptional inhibitors following acute resistance exercise.

    The levels of the Smad inhibitors SKI and active Notch were determined at the end of a 20 minute bout of high force lengthening contractions and then 0.5, 3, 6, 18, and 48 hours later. SKI and Notch protein was normalized to GAPDH.

    Rat muscles were powdered on dry ice using a mortar and pestle and polytron homogenized in 10-fold mass excess of ice cold sucrose lysis buffer and 0.1% DTT. This was vortexed for 30 minutes at 4°C and centrifuged at 4°C for 10 minutes at 10,000×g to remove insoluble material.

    Equal aliquots of protein were diluted in Laemmli sample buffer and boiled for 5 minutes. 5–10 µg of sample was then subjected to SDS-PAGE on 10% acrylamide gels at a constant current equal to 20 mA per gel and transferred to Protran nitrocellulose membrane using a semidry transfer apparatus at 100 V for 1 hour. Membranes were blocked in 5% dry milk in TBST, and then incubated over night at 4°C with ab8925 in TBST at 1:1,000.

    The membranes were then washed 3x in TBST before incubation for 1 hour at room temperature with peroxidase-conjugated secondary antibodies in TBST at 1:10,000. ECL detection.

  • ab8925 staining activated Notch 1 in murine dermis (including follicle) by Immunohistochemistry (Frozen sections).

    Tissue was fixed with paraformaldehyde and permeabilized using 0.1% Triton. Samples were then blocked with 10% serum for 1 hour at 19°C followed by incubation with the primary antibody at a 1/100 dilution for 16 hours at 4°C. An Alexa Fluor® conjugated donkey anti-rabbit polyclonal was used as secondary antibody at a 1/200 dilution.

    Counterstain DAPI (blue).

    See Abreview

  • ab8925 at a 1:500 dilution staining activated Notch1 in human ovarian carcinoma using an automated system (DAKO Autostainer Plus).

    Using this protocol there is strong staining of activated Notch 1 in nuclear/nucleolar compartments of the ovarian cortex.
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH 6.1 in a DAKO PT link. Slides were blocked in 3% H2O2/methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes.

    Slides were counterstained with hematoxylin and coverslipped under DePeX.

    Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

  • Rabbit polyclonal (ab8925) at 1/500, against myc-tagged transiently transfected mouse Notch constructs in HEK-293 (Human epithelial cell line from embryonic kidney) cells.

    Lane M: Mol wt markers
    Lane 1 No transfection
    Lane 2 N1 (mouse deleted extracellular domain)-myc
    Lane 3 N1 (mouse intracellular domain)-myc
    Lane 4 N2 (mouse deleted extracellular domain)-myc
    Lane 5 N2 (mouse intracellular domain)-myc

    Lane 6 N3 (mouse deleted extracellular domain)-myc
    Lane 7 N3 (mouse intracellular domain)-myc
    Lane 8 N4 (mouse deleted extracellular domain)-myc
    Lane 9 N4 (mouse intracellular domain)-myc
    Lane 10 N1 (mouse deleted extracellular domain)(V to G)-myc

  • ab8925 staining actived Notch1 in the NIH/3T3 (Mouse embryo fibroblast cell line) cells by ICC/IF (Immunocytochemistry/immunofluorescence).

    Cells were fixed with paraformaldehyde, permeabilized with Triton X-100 0.1% and blocked with 10% serum for 30 minutes at 24°C. Samples were incubated with primary antibody (1/100) for 16 hours at 4°C. An Alexa Fluor®488-conjugated Donkey anti-rabbit IgG polyclonal(1/500) was used as the secondary antibody.

    See Abreview

References

This product has been referenced in:
  • Lu Y  et al. Effect of midkine on gemcitabine resistance in biliary tract cancer. Int J Mol Med 41:2003-2011 (2018). Read more (PubMed: 29344648) »
  • Xiong S  et al. Cancer-associated fibroblasts promote stem cell-like properties of hepatocellular carcinoma cells through IL-6/STAT3/Notch signaling. Am J Cancer Res 8:302-316 (2018). Read more (PubMed: 29511600) »
See all 149 Publications for this product

Customer reviews and Q&As

1-10 of 74 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (mcf7)
Specification
mcf7
Fixative
acetone/methanol 50/50
Permeabilization
No
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Apr 06 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - nuclear (MCF-7)
Loading amount
25 µg
Specification
MCF-7
Treatment
Mock or EDTA 5mM for 10mins
Gel Running Conditions
Reduced Denaturing (8-12% gradient)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Mar 27 2013

Answer

Thank you for your enquiry.

I can confirm that ab8925 antibody is sold as whole antiserum. Unpurified antibodies, such as those sold as whole antiserum will not have a concentration stated on the datasheet. Antibody concentration is usually determined by protein assay, and serum / ascites / tissue culture supernatant will contain a lot of other proteins, which means the antibody quantification would not be accurate.

I can confirm that for whole antiserum, concentration of antibody is known to very between 1 - 10 mg/ml.

I am sorry we are not able to provide an exact concentration on this occasion, but hope this information will be helpful to you. If you have any further questions, please do not hesitate to contact us.

Read More

Answer

Thank you for contacting Abcam and for your interest in our products.


Regarding the activated Notch1 antibody ab8925, this antibody is validated and guaranteed to work in IHC (frozen and paraffin sections), ICC, WB, and Flow cytometry using mouse and human samples.


If you are using the antibody in one of these applications with either of these species than if the product does not work, you will receive a replacement or credit when contacted within 6 months of purchase. Please see our Abpromise on our website: https://www.abcam.com/abpromise.


If you are using the antibody in an untested application or species you may be eligible for a testing discount. Please reply to this email with information regarding the untested species and/or application you wish to test and I would be happy to assist you. You can also find additional information regarding this testing program on our website: https://www.abcam.com/collaborationdiscount.


I hope this information is helpful. Please do not hesitate to contact us if you have any additional questions or concerns.

Read More
Application
Western blot
Sample
Mouse Cell lysate - whole cell (MEF cells)
Gel Running Conditions
Reduced Denaturing (4-12% Gradient Gel)
Loading amount
20 µg
Specification
MEF cells
Blocking step
Licor Blocking Buffer as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Dec 11 2017

Application
Western blot
Sample
Mouse Tissue lysate - other (Brain- membrane and cytosolic fractions)
Gel Running Conditions
Reduced Denaturing (4-12% gradient gel)
Loading amount
10 µg
Specification
Brain- membrane and cytosolic fractions
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Jul 26 2017

Application
Western blot
Sample
Mouse Tissue lysate - other (brain)
Gel Running Conditions
Non-reduced Denaturing (10% acrylamide)
Loading amount
25 µg
Treatment
25 uM DAPT
Specification
brain
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Ms. Victoria Lladó

Verified customer

Submitted Oct 16 2015

Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (hepatocytes)
Permeabilization
Yes - 0.1% Triton X-100 in TBS for 5-10 minutes
Specification
hepatocytes
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 23°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Sep 11 2015

Application
IHC - Wholemount
Sample
Zebrafish Embryo (48hpf)
Specification
48hpf

Abcam user community

Verified customer

Submitted Mar 23 2015

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
Dakocytomation X0909 as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: RT°C
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate Buffer pH6.0
Sample
Mouse Tissue sections (human melanoma cell line injected s.c. in mouse.)
Specification
human melanoma cell line injected s.c. in mouse.
Permeabilization
Yes - 0.25%Triton-X100
Fixative
Formaldehyde

Hongwei Shao

Verified customer

Submitted May 02 2014

1-10 of 74 Abreviews or Q&A

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