Preparation And Storage
Recommended Assay Procedures
Bioimaging
1. Seed the cells in appropriate culture medium at ~10,000 cells per well in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219) and culture overnight.
2. Remove the culture medium from the wells, and fix the cells by adding 100 μl of BD Cytofix™ Fixation Buffer (Cat. No. 554655) to each well. Incubate for 10 minutes at room temperature (RT).
3. Remove the fixative from the wells, and permeabilize the cells using either BD Perm Buffer III, 90% methnaol, or Triton™ X-100:
a. Add 100 μl of -20°C 90% methanol or Perm Buffer III (Cat. No. 558050) to each well and incubate for 5 minutes at RT.
OR
b. Add 100 μl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.
4. Remove the permeabilization buffer, and wash the wells twice with 100 μl of 1× PBS.
5. Remove the PBS, and block the cells by adding 100 μl of BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) to each well. Incubate for 30 minutes at RT.
6. Remove the blocking buffer and add 50 μl of the optimally titrated primary antibody (diluted in Stain Buffer) to each well, and incubate for 1 hour at RT.
7. Remove the primary antibody, and wash the wells three times with 100 μl of 1× PBS.
8. Remove the PBS, and add the second step reagent at its optimally titrated concentration in 50 μl to each well, and incubate in the dark for 1 hour at RT.
9. Remove the second step reagent, and wash the wells three times with 100 μl of 1× PBS.
10. Remove the PBS, and counter-stain the nuclei by adding 200 μl per well of 2 μg/ml Hoechst 33342 (e.g., Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.
11. View and analyze the cells on an appropriate imaging instrument.
Bioimaging: For more detailed information please refer to http://www.bdbiosciences.com/support/resources/protocols/ceritifed_reagents.jsp
Western blot: For more detailed information please refer to http://www.bdbiosciences.com/pharmingen/protocols/Western_Blotting.shtml
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Triton is a trademark of the Dow Chemical Company.
Companion Products
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During the cell cycle, most eukaryotic cells double in mass, replicate their DNA and then distribute identical copies of their genome to progeny cells during mitosis. An internal biochemical clock ensures that all the necessary events occur in proper sequence and at the appropriate time. Cell in M phase contain a dominant regulatory factor known as maturation promoting factor (MPF). MPF triggers a variety of enzymatic and ultrastructural changes that are necessary for cell division. In higher eukaryotes, these mitosis-specific alterations include disassembly of the nuclear envelope, packaging of the DNA into chromosomes and assembly of the mitotic spindle. Purified MPF is a regulator of a protein kinase cascade and is evolutionarily conserved in all eukaryotic cells ranging from yeast to man. MPF consists predominantly of two polypeptides, cyclin B1 and p34, and contains protein kinase activity itself. It is the major M-phase-specific histone H1 kinase, but also phosphorylates a variety of other substrates including lamins, nucleolin, RNA polymerase II, retinoblastoma protein, SV40 large T antigen, p53, and the oncogenes c-src and c-abl. Cyclin B1 migrates at a reduced molecular weight of 62 kDa on SDS-PAGE.
Development References (8)
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Dunphy WG, Brizuela L, Beach D, Newport J. The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis. Cell. 1988; 54(3):423-431. (Biology). View Reference
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Gong J, Traganos F, Darzynkiewicz Z. Simultaneous analysis of cell cycle kinetics at two different DNA ploidy levels based on DNA content and cyclin B measurements. Cancer Res. 1993; 53(21):5096-5099. (Clone-specific: Flow cytometry, Immunofluorescence).
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Gong J, Traganos F, and Darzynkiewicz Z. Expression of cyclins B and E in individual MOLT-4 cells and in stimulated human lymphocytes during progression through the cell cycle. Int J Oncol. 1993; 3:1037-1042. (Clone-specific: Flow cytometry, Immunofluorescence).
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Hoffmann I, Clarke PR, Marcote MJ, Karsenti E, Draetta G. Phosphorylation and activation of human cdc25-C by cdc2--cyclin B and its involvement in the self-amplification of MPF at mitosis. EMBO J. 1993; 12(1):53-63. (Biology). View Reference
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Jessus C, Beach D. Oscillation of MPF is accompanied by periodic association between cdc25 and cdc2-cyclin B. Cell. 1992; 68(2):323-332. (Biology). View Reference
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Keyomarsi K, Pardee AB. Redundant cyclin overexpression and gene amplification in breast cancer cells. Proc Natl Acad Sci U S A. 1993; 90(3):1112-1116. (Clone-specific: Western blot). View Reference
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Pines J, Hunter T. Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport. J Cell Biol. 1991; 115(1):1-17. (Clone-specific: Fluorescence microscopy). View Reference
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Zhang H, Xiong Y, Beach D. Proliferating cell nuclear antigen and p21 are components of multiple cell cycle kinase complexes. Mol Biol Cell. 1993; 4(9):897-906. (Clone-specific: Immunoprecipitation). View Reference
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product as described.
Comparisons, where applicable, are made against older BD technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
