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
The p53 protein is critical to regulation of normal cell growth and is a suppressor of tumor cell proliferation. Inactivation of p53 by a number of mechanisms, such as missense mutations or interaction with oncogenic viral or cellular proteins, can result in tumor progression. In addition, Bcl2 and p53 are involved in apoptosis in an antagonistic fashion such that overexpressed Bcl2 inhibits p53-induced apoptosis. 53BP1 and 53BP2 were identified in a yeast two-hybrid screen of proteins that bind p53. Both 53BP1 and 53BP2 bind wild type p53, but not mutant p53 found in tumor cells. p53BP1 is localized to the cytoplasm and nucleus, while p53BP2 is found only in the cytoplasm. 53BP1 has BRCT motifs found in proteins involved in cell cycle control and DNA repair. DNA damage leads to 53BP1 hyperphosphorylation, which may be mediated by ATM. 53BP2 has four ankyrin repeats and a SH3 domain that are required for interactions with Bcl2 and p53. Overexpression of 53BP2 in 293 cells inhibits progression of the cell cycle in G2/M phase, while co-transfection of 53BP2 with p53 in H358 cells enhances p53-mediated transcriptional activation. The interaction between 53BP2 and p53 may be regulated by Bcl2, since competition experiments demonstrate that Bcl2 prevents p53 binding to 53BP2. In addition, 53BP2 can also bind the apoptotic-related p65 subunit of NFκB and this subunit can inhibit 53BP2-induced cell death.
Development References (3)
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Iwabuchi K, Bartel PL, Li B, Marraccino R, Fields S. Two cellular proteins that bind to wild-type but not mutant p53. Proc Natl Acad Sci U S A. 1994; 91(13):6098-6102. (Biology). View Reference
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Iwabuchi K, Li B, Massa HF, Trask BJ, Date T, Fields S. Stimulation of p53-mediated transcriptional activation by the p53-binding proteins, 53BP1 and 53BP2. J Biol Chem. 1998; 273(40):26061-26068. (Biology). View Reference
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Rappold I, Iwabuchi K, Date T, Chen J. Tumor suppressor p53 binding protein 1 (53BP1) is involved in DNA damage-signaling pathways. J Cell Biol. 2001; 153(3):613-620. (Biology). View Reference
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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.
