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Synthetic peptide conjugated to KLH derived from within residues 1200 - 1300 of Human Ki67.
(Peptide available as ab15581.)
Our Abpromise guarantee covers the use of ab15580 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC - Wholemount||Use at an assay dependent concentration.|
|IHC-P||Use a concentration of 0.1 - 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|IHC-FrFl||1/500. (see Abreview)|
|Flow Cyt||1/100. ab171870-Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.|
|IHC-Fr||1/100 - 1/1000.|
|ICC/IF||Use a concentration of 1 µg/ml.|
|ICC||Use at an assay dependent concentration.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 345, 395 kDa (predicted molecular weight: 359 kDa).Can be blocked with Ki67 peptide (ab15581).
Higher molecular weight proteins like ki67 may be more difficult to detect in WB. We recommend several potential optimisation steps: loading higher amounts of protein (20 ug and above), using lower percentage gels and/or Tris-Acetate gels, increasing antioxidant to maintain protein reduction, decreasing methanol and increasing SDS in the transfer buffer and increasing time and voltage of transfer. Larger proteins can be subject to degradation more than smaller proteins so lower molecular weight bands may be present. For further information or support please contact our Scientific Support Team.
|IHC-FoFr||Use at an assay dependent concentration.|
ab15580 staining Ki67 in wild-type HAP1 cells (top panel) and Ki67 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab15580 at 1μg/ml concentration and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
IHC image of ab15580 staining in mouse spleen formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol B. 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 ab15580, 5µg/ml, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
IHC image of Ki67 staining in human spleen formalin fixed paraffin embedded tissue section, performed on a Leica Bond 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 ab15580, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chrogen. 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.
Fluorescent confocal microscopy (20x) of mouse (P0) olfactory bulb, outer glomeruli layer, showing Ki67 immunoreactivity (ab15580; 1/1000; overnight at RT, 0.25% TX-100 no blocking step) using a secondary goat anti-rabbit fluorescent antibody (Alexa Fluor 488;1/300 2h at RT.
SK-N-SH cells were permitted to grow to confluency, then serum starved for 48 hours and predominantly driven into G0. The cells were then paraformaldehyde fixed and immunofluorescently labelled with anti-Ki67 (ab15580) at a dilution of 1/1000. The majority of the cells show little or no Ki67 staining, indicating they are in G0 arrest (red cells). Two cells however show strong nucleolar Ki67 staining indicating they are still cycling (green cells). The DNA is stained with DAPI and is shown in red. The Ki67 staining is shown in green. x 63 magnification.
Similar results were seen with an asynchronous population of HeLa cells. The Ki67 staining was localised to the periphery of the nucleoli and throughout the nucleoplasm of proliferating cells. (This data is not shown but is available upon request).
Immunohistochemistry analysis of Formaldehyde-fixed paraffin-embedded mouse tumour tissue sections labelling Ki67 with ab15580 at 1/2000. The secondary antibody was biotin conjugated goat polyclonal vector at a dilution of 1/250.
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