The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
1/2000 - 1/10000. Detects a band of approximately 100 kDa (predicted molecular weight: 100 kDa).
Use at 2-5 µg/mg of lysate.
Mitotic kinesin required for chromosome passenger complex (CPC)-mediated cytokinesis. Following phosphorylation by PLK1, involved in recruitment of PLK1 to the central spindle. Interacts with guanosine triphosphate (GTP)-bound forms of RAB6A and RAB6B. May act as a motor required for the retrograde RAB6 regulated transport of Golgi membranes and associated vesicles along microtubules. Has a microtubule plus end-directed motility.
Belongs to the kinesin-like protein family. Contains 1 kinesin-motor domain.
Phosphorylated by PLK1 at Ser-528 during mitosis, creating a docking site for PLK1 and recruiting PLK1 at central spindle.
IHC image of KIF20A staining in Human skin melanoma 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 ab70791, 5µg/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.
Western blot - Anti-KIF20A antibody (ab70791)
All lanes : Anti-KIF20A antibody (ab70791) at 0.1 µg/ml
Lane 1 : HeLa whole cell lysate at 50 µg Lane 2 : HeLa whole cell lysate at 15 µg Lane 3 : HeLa whole cell lysate at 5 µg Lane 4 : 293T whole cell lysate at 50 µg
Developed using the ECL technique.
Predicted band size: 100 kDa Observed band size: 100 kDa Additional bands at: 120 kDa, 31 kDa, 60 kDa, 68 kDa, 73 kDa. We are unsure as to the identity of these extra bands.
HeLa whole cell lysate (1 mg for IP, 20% of IP loaded) with ab70791 at 1 µg/ml for WB, subsequent to using for IP at 3 µg/mg lysate. Lane 2: IgG control. Detection: Chemiluminescence with an exposure time of 10 seconds.