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.
|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.