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Synthetic peptide corresponding to Human NOTCH3 aa 2300 to the C-terminus (C terminal) conjugated to Keyhole Limpet Haemocyanin (KLH).
(Peptide available as
Our Abpromise guarantee covers the use of ab23426 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 97,280 kDa (predicted molecular weight: 244 kDa).|
|IHC-P||Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. PubMed: 19965627|
|ICC/IF||Use at an assay dependent concentration. PubMed: 20680961|
|IHC-FoFr||Use at an assay dependent concentration.|
ab23426 staining NOTCH3 in mouse pancreatic cancer tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with acetone and blocked with 10% serum for 1 hour at 20°C. Samples were incubated with the primary antibody (1/100 in PBS) for 8 hours at 4°C. A biotin-conjugated goat anti-rabbit IgG polyclonal (1/1000) was used as the secondary antibody.
This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Bovine Serum Albumin before being incubated with ab23426 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.
The band observed at 97 kDa is thought to correspond to the notch-derived peptide containing the intracellular domain (NICD) of NOTCH3 as described in the literature (PMID:10712431).
ICC/IF image of ab23426 stained MCF7 cells. The cells were 100% methanol fixed (5 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 (ab23426, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit 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.
ab23426 staining NOTCH3 in human breast cancer tissue sections by immunohistochemistry (Formalin/PFA-fixed paraffin embedded sections). Tissue underwent fixation in paraformaldehyde, heat-mediated antigen retrieval in citrate buffer pH6.0 and blocking for 15 minutes at 20°C (5 minutes for peroxidase blocking and 10 minutes for protein blocks). The primary antibody was diluted 1/250 and incubated with sample for 45 minutes at 20°C. A HRP-conjugated goat polyclonal to rabbit IgG was used undiluted as secondary.
ab14404 staining NOTCH3 in the COS1 fibroblast cell line from Monkey Kkidney by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with Triton X-100 0.1% in PBS and blocked with 1% BSA for 30 minutes at 25°C. Samples were incubated with primary antibody (1/200) for 16 hour at 4°C. An Alexa Fluor® 488-conjugated Goat anti-rabbit polyclonal (1/500) was used as the secondary antibody.