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Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human p16 ARC.
Our Abpromise guarantee covers the use of ab118459 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.|
|IHC-P||Use a concentration of 1 µg/ml.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 18 kDa (predicted molecular weight: 16 kDa).|
Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: p16 ARC knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: HT29 whole cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab118459 observed at 16 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab118459 was shown to recognize p16 ARC when p16 ARC knockout samples were used, along with additional cross-reactive bands. Wild-type and p16 ARC knockout samples were subjected to SDS-PAGE. Ab118459 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1 µg/ml and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.
IHC image of p16 ARC staining in Human lung adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica 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 ab118459, 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.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
ICC/IF image of ab118459 stained MCF-7 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 ab118459 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 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. This antibody also gave a positive result in formaldehyde fixed (4%, 10min) HeLa, Hek293, and HepG2 cell types, and in Methanol fixed (100%, 5min) HeLa, and HepG2 cell types.
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