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
Overview
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Product name
Anti-activated Notch1 antibody
See all activated Notch1 primary antibodies -
Description
Rabbit polyclonal to activated Notch1 -
Host species
Rabbit -
Tested Applications & Species
See all applications and species dataApplication Species IHC-P HumanWB Recombinant fragment
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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 asab730) -
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
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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
Images
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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.
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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.
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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)-mycLane 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
