![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](http://www.bioprodhub.com/system/product_images/ab_products/2/sub_4/1435_ab65297-205648-IPVI002ab6529720mMod.jpg)
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
All lanes : Anti-Notch1 antibody (ab65297) at 1 µg/mlLane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 2 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate Lane 3 : HEK293 (Human embryonic kidney cell line) Whole Cell LysateLane 4 : SK N SH (Human neuroblastoma) Whole Cell Lysate Lane 5 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate Lysates/proteins at 10 µg per lane.SecondaryGoat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilutionPerformed under reducing conditions.
IHC image of Notch1 staining in Human Hippocampus FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab65297, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX
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).See Abreview
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).See Abreview