Immunofluorescent analysis of Ki-67 was performed on HeLa cells serum-starved for about 16 hours and then serum released for 4 hours. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0. 25% of Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with ABfinity™ Ki-67 recombinant rabbit monoclonal antibody (Product # 701198) at a dilution of 1:400 in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Flour® 488 goat anti-rabbit IgG secondary antibody (Product # A11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 594 phalloidin (Product # A12381). Panel d is a merged image showing nuclear localization. The images were captured using a Nikon microscope at 20X magnification.
|Tested species reactivity||Human|
|Published species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Peptide corresponding to amino acids 1213–1232 of human Ki-67|
|Contains||0.09% sodium azide|
|Storage Conditions||Maintain refrigerated at 2-8°C for up to 1 month. For long term storage store at -20°C|
|Tested Applications||Dilution *|
|ChIP assay (ChIP)||1 ul|
|Flow Cytometry (Flow)||1:100-1:500|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
|Immunohistochemistry (Paraffin, Frozen) (IHC (P, F))||See 1 publications below|
This antibody is predicted to react with non-human primate, mouse and rat based on sequence homology.
ABfinity™ recombinant antibodies are rabbit monoclonal antibodies, unmatched for producing superior results. ABfinity™ antibodies are developed by immunizing animals, screening for functionality, cloning the immunogen-specific antibody genes into high-level mammalian expression vectors, produced on a large scale, and purified with Protein A.
ABfinity™ monoclonal antibodies resemble rabbit monoclonals isolated from serum or produced by hybridomas, but demonstrate greater specificity and sensitivity. Because ABfinity™ recombinant antibodies are derived from cloned DNA sequences of the heavy and light antibody chains, they are not susceptible to cell-line drift or lot-to-lot variation, thus allowing for peak specificity and performance.
Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain.
Ki-67 is a nuclear protein that is expressed during various stages in the cell cycle, particularly during late G1, S, G2, and M phases. The protein has a forkhead associated domain (FHA) through which it associates with the euchromatin at the perichromosomal layer with centromeric heterochromatin, and with the nucleolus. Ki-67 is shown to have a cell cycle dependent topographical distribution, with perinucleolar expression at G1, expression in the nuclear matrix at G2, and expression on the chromosomes during M phase. Ki-67 is commonly used as a proliferation marker because it is not detected in G0 cells, but increases steadily from G1 through mitosis.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
|Human||Not Cited||Orthokeratinized odontogenic cyst: a clinicopathologic study of 61 cases.||Dong Q,Pan S,Sun LS,Li TJ||Archives of pathology and laboratory medicine (134:271)||2010|