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Synthetic peptide conjugated to KLH derived from within residues 2500 to the C-terminus of Human Notch1.
(Peptide available as ab68580.)
Our Abpromise guarantee covers the use of ab65297 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||Use a concentration of 5 µg/ml.|
|IP||Use a concentration of 5 µg/ml.|
|IHC-P||Use a concentration of 1 µg/ml.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 120 kDa (predicted molecular weight: 272 kDa).|
|Flow Cyt||Use at an assay dependent concentration. PubMed: 24136221ab171870-Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.|
Notch1 was immunoprecipitated using 0.5mg Hek293 whole cell extract, 5µg of Rabbit polyclonal to Notch1 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hek293 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab65297.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 120kDa; Notch1
ICC/IF image of ab65297 stained HepG2 cells. The cells were 4% formaldehyde fixed (10 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 ab65297 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) 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.
ab65297 staining Notch1 (green) in Mouse embryo tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 5% serum for 1 hour at room temperature; antigen retrieval was by heat mediation in a 0.5% citrate buffer. Samples were incubated with primary antibody (1/50 in 5% Goat serum) for 2 hours. An Alexa Fluor® 488-conjugated Goat anti-rabbit IgG polyclonal (1/300) was used as the secondary antibody. Red - Myh6/7, Blue - nuclei (DAPI).
ab65297 staining Notch1 (green) in Mouse embryo tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with paraformaldehyde, permeabilized with 1% Triton X-100 and blocked with 5% serum for 2 hours at room temperature. Samples were incubated with primary antibody (1/100 in 5% Goat serum) for 2 hours. An Alexa Fluor® 488-conjugated Goat anti-rabbit IgG polyclonal (1/400) was used as the secondary antibody. Red - Myh6/7, Blue - nuclei (DAPI).