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Anti-activated Notch1 antibody (ab8925)

  • Datasheet
  • SDS
Reviews (23)Q&A (51)References (214)

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Western blot - Anti-activated Notch1 antibody (ab8925)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-activated Notch1 antibody (ab8925)
  • Western blot - Anti-activated Notch1 antibody (ab8925)

Key features and details

  • Rabbit polyclonal to activated Notch1
  • Suitable for: IHC-P, WB
  • Reacts with: Mouse, Human
  • Isotype: IgG

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Overview

  • Product name

    Anti-activated Notch1 antibody
    See all activated Notch1 primary antibodies
  • Description

    Rabbit polyclonal to activated Notch1
  • Host species

    Rabbit
  • Tested Applications & Species

    Application Species
    IHC-P
    Human
    WB
    Recombinant fragment
    See all applications and species data
  • 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)
    Run BLAST with BLAST the sequence with ExPASy Run BLAST with BLAST the sequence with NCBI
  • 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

  • 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: 0.1% Sodium azide
    Constituents: 0.4% Potassium phosphate, 0.87% Sodium chloride
  • Concentration information loading...
  • Purity

    Whole antiserum
  • Purification notes

    Label may show concentration. This is total protein concentration. Total and specific IgG concentration have not been determined.
  • Primary antibody 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.
  • Clonality

    Polyclonal
  • Isotype

    IgG
  • Research areas

    • Neuroscience
    • Neurology process
    • Notch Pathway
    • Epigenetics and Nuclear Signaling
    • Transcription
    • Other factors
    • Stem Cells
    • Signaling Pathways
    • Notch
    • Surface Molecules
    • Stem Cells
    • Signaling Pathways
    • Notch
    • Nuclear
    • Stem Cells
    • Neural Stem Cells
    • Intracellular
    • Stem Cells
    • Neural Stem Cells
    • Surface Molecules
    • Stem Cells
    • Hematopoietic Progenitors
    • Surface Molecules
    • Cardiovascular
    • Heart
    • Cardiogenesis
    • Transcription factors/regulators
    • Stem Cells
    • Neural Stem Cells
    • Neural Crest Stem Cells
    • Developmental Biology
    • Embryogenesis
    • Somatogenesis

Associated products

  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
  • Immunizing Peptide (Blocking)

    • Human activated Notch1 peptide (ab730)
  • Isotype control

    • Rabbit IgG, polyclonal - Isotype Control (ChIP Grade) (ab171870)

Applications

The Abpromise guarantee

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.

Guaranteed

Tested applications are guaranteed to work and covered by our Abpromise guarantee.

Predicted

Predicted to work for this combination of applications and species but not guaranteed.

Incompatible

Does not work for this combination of applications and species.

Application Species
IHC-P
Human
WB
Recombinant fragment
All applications
Rat
Application Abreviews Notes
IHC-P (3)
1/200.
WB (8)
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.
Notes
IHC-P
1/200.
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.

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.
  • Target information above from: UniProt accession P46531 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 4851 Human
    • Entrez Gene: 18128 Mouse
    • Entrez Gene: 25496 Rat
    • Omim: 190198 Human
    • SwissProt: P46531 Human
    • SwissProt: Q01705 Mouse
    • SwissProt: Q07008 Rat
    • Unigene: 495473 Human
    • Unigene: 290610 Mouse
    • Unigene: 25046 Rat
    see all
  • 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

  • Western blot - Anti-activated Notch1 antibody (ab8925)
    Western blot - Anti-activated Notch1 antibody (ab8925)MacKenzie et al PLoS One. 2013 Jul 2;8(7):e68743. doi: 10.1371/journal.pone.0068743. Print 2013. Fig 5. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    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.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-activated Notch1 antibody (ab8925)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-activated Notch1 antibody (ab8925)

    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.

  • Western blot - Anti-activated Notch1 antibody (ab8925)
    Western blot - Anti-activated Notch1 antibody (ab8925)This image is courtesy of Stacey Huppert, Ma. Xenia U. Garcia (lab of Raphael Kopan)

    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

Protocols

  • Immunohistochemistry protocols
  • Immunocytochemistry & immunofluorescence protocols
  • Western blot protocols

Click here to view the general protocols

Datasheets and documents

    • Datasheet
    • SDS
  • References (214)

    Publishing research using ab8925? Please let us know so that we can cite the reference in this datasheet.

    ab8925 has been referenced in 214 publications.

    • Duhachek-Muggy S  et al. Radiation mitigation of the intestinal acute radiation injury in mice by 1-[(4-nitrophenyl)sulfonyl]-4-phenylpiperazine. Stem Cells Transl Med 9:106-119 (2020). PubMed: 31464098
    • Zhu M  et al. Effects of bisphenol A and its alternative bisphenol F on Notch signaling and intestinal development: A novel signaling by which bisphenols disrupt vertebrate development. Environ Pollut 263:114443 (2020). PubMed: 32311622
    • Kaminska A  et al. Disruption of androgen signaling during puberty affects Notch pathway in rat seminiferous epithelium. Reprod Biol Endocrinol 18:30 (2020). PubMed: 32299422
    • Jiang N  et al. HIF-1?-regulated miR-1275 maintains stem cell-like phenotypes and promotes the progression of LUAD by simultaneously activating Wnt/ß-catenin and Notch signaling. Theranostics 10:2553-2570 (2020). PubMed: 32194819
    • Wei B  et al. MicroRNA-145-5p attenuates high glucose-induced apoptosis by targeting the Notch signaling pathway in podocytes. Exp Ther Med 19:1915-1924 (2020). PubMed: 32104249
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
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    1-10 of 74 Abreviews or Q&A

    Immunocytochemistry/ Immunofluorescence abreview for Anti-activated Notch1 antibody

    Average
    Abreviews
    Abreviews
    abreview image
    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
    Read More

    Abcam user community

    Verified customer

    Submitted Apr 06 2013

    Western blot abreview for Anti-activated Notch1 antibody

    Average
    Abreviews
    Abreviews
    abreview image
    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
    Read More

    Abcam user community

    Verified customer

    Submitted Mar 27 2013

    Question

    Product code: 8925
    Lot number: 759251
    Inquiry: Hi, I am using the ab8925 antibody for IHC. I want to use as negative control the rabbit isotype IgG. Could you please specify the concentration of IgG for this antibody? I am using the lot number: 759251 and the antibody concentration is 80mg/ml. Regards,

    Read More

    Abcam community

    Verified customer

    Asked on Jul 31 2012

    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

    Abcam Scientific Support

    Answered on Jul 31 2012

    Question

    Our lab is looking to order ab8925 (activated Notch 1 antibody).
    We requested for a free aliquot but was told that it's not your company's policy to do that. It was suggested that I ask about testing discount program to Scientific Support.
    Please let me know how I can go forward with this.
    Thanks very much,

    Read More

    Abcam community

    Verified customer

    Asked on Mar 23 2012

    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

    Abcam Scientific Support

    Answered on Mar 23 2012

    Western blot abreview for Anti-activated Notch1 antibody

    Excellent
    Abreviews
    Abreviews
    abreview image
    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
    Read More

    Abcam user community

    Verified customer

    Submitted Dec 11 2017

    Western blot abreview for Anti-activated Notch1 antibody

    Poor
    Abreviews
    Abreviews
    abreview image
    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
    Read More

    Abcam user community

    Verified customer

    Submitted Jul 26 2017

    Western blot abreview for Anti-activated Notch1 antibody

    Poor
    Abreviews
    Abreviews
    abreview image
    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
    Read More

    Ms. Victoria Lladó

    Verified customer

    Submitted Oct 16 2015

    Immunocytochemistry/ Immunofluorescence abreview for Anti-activated Notch1 antibody

    Good
    Abreviews
    Abreviews
    abreview image
    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
    Read More

    Abcam user community

    Verified customer

    Submitted Sep 11 2015

    IHC - Wholemount abreview for Anti-activated Notch1 antibody

    Average
    Abreviews
    Abreviews
    abreview image
    Application
    IHC - Wholemount
    Sample
    Zebrafish Embryo (48hpf)
    Specification
    48hpf
    Read More

    Abcam user community

    Verified customer

    Submitted Mar 23 2015

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-activated Notch1 antibody

    Good
    Abreviews
    Abreviews
    abreview image
    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
    Read More

    Hongwei Shao

    Verified customer

    Submitted May 02 2014

    1-10 of 74 Abreviews or Q&A

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