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% methanol, 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/support/resources/protocols/monoclonal_anti.jsp
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- 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.
- Triton is a trademark of the Dow Chemical Company.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
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Cyclin-dependent kinase 2 (Cdk2) is a member of a family of cdc2-related cell cycle protein kinases. Cdk2 shares 60% identity with cdc2 and its activity is regulated by phosphorylation in a similar fashion. Cdk2 is expressed earlier in the cell cycle than is cdc2. Like p34 [cdc2], p33 [cdk2] associates with Cyclin A in human cells. However, kinase activity associated with Cyclin A-Cdk2 is present in S phase, whereas, the kinase activity associated with Cyclin A-cdc2 is found only in G2. Cdk2 can also complex with cyclins E, D1, and D3. It is not known if the D cyclins can form active complexes with Cdk2. Cyclin E-Cdk2 kinase is active in the G1 and S phases of the cell cycle and is important (as is Cyclin A-Cdk2) for the progression from G1 to S phase. The levels of Cyclin A-Cdk2 are maximal at the G1/S transition and both Cdk2 and Cyclin A associate with DNA in the initiation complex during replication. The Rb protein has been identified as a substrate for Cdk2-Cyclin E and/or Cdk2-Cyclin A in vivo. This observation is supported by further evidence which shows that Cdk2 is activated and specifically localized to the nucleus during late G1, S phase, and G2.
Development References (5)
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Hinds PW, Mittnacht S, Dulic V, Arnold A, Reed SI, Weinberg RA. Regulation of retinoblastoma protein functions by ectopic expression of human cyclins. Cell. 1992; 70(6):993-1006. (Biology). View Reference
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Mal A, Chattopadhyay D, Ghosh MK, Poon RY, Hunter T, Harter ML. p21 and retinoblastoma protein control the absence of DNA replication in terminally differentiated muscle cells. J Cell Biol. 2000; 149(2):281-292. (Biology: Immunoprecipitation, In vitro kinase assay, Western blot). View Reference
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Mohapatra S, Agrawal D, Pledger WJ. p27Kip1 regulates T cell proliferation. J Biol Chem. 2001; 276(24):21976-21983. (Biology: Western blot). View Reference
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Porter DC, Zhang N, Danes C, et al. Tumor-specific proteolytic processing of cyclin E generates hyperactive lower-molecular-weight forms. Mol Cell Biol. 2001; 21(18):6254-6269. (Biology: Western blot). View Reference
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Saitoh H, Pizzi MD, Wang J. Perturbation of SUMOlation enzyme Ubc9 by distinct domain within nucleoporin RanBP2/Nup358. J Biol Chem. 2002; 277(7):4755-4763. (Biology: Immunofluorescence). 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.
