Preparation and Storage
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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/pharmingen/protocols/Western_Blotting.shtml
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.
- 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.
関連製品
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MDM2, originally described as a gene product of mouse double minute chromosomes, is a nuclear oncoprotein that can inhibit the action of certain tumor suppressor proteins. For example, MDM2 binds to the acidic activation domain (residues 2042) of the p53 tumor suppressor protein, and the p53-MDM2 complex down regulates the transcriptional activity of p53. p53 plays a role in the normal cell cycle by activating the transcription of genes that cause arrest in G1. The expression of MDM2 is itself, induced by p53 and may be a way for p53 to self-regulate its activity during the normal cell cycle. However, overexpression of MDM2 results in the loss of p53-regulated growth control and consequently, deregulated cell proliferation. MDM2 also binds to the Retinoblastoma tumor suppressor protein (Rb) and inhibits its growth regulatory function. MDM2 can directly augment proliferation by binding to two transcription factors E2F1 and DP1, and stimulating the activity of the S-phase inducing E2F1/DP1 heterodimer. MDM2 migrates at a reduced molecular weight of ~95 kDa.
The SMP14 clone has been reported to recognize human, mouse and rat MDM2 while exhibiting a slight cross-reactivity with cytokeratins 6, 14 and 16 in some experimental systems. In the immunoprecipitation application, SMP14 has been reported to precipitate MDM2 and p53-MDM2 complexes. MCF7 human breast carcinoma cells (ATCC HTB-22) and NIH/3T3 mouse fibroblasts (ATCC CRL-1658) are suggested as western blot and immunoprecipitation positive controls. SMP14 has been reported to be useful for the immunohistochemical staining of acetone-fixed, frozen sections and of formalin-fixed, paraffin-embedded tissue sections. In addition to a nuclear staining of MDM2, cytoplasmic staining may also be observed which is likely to be atrributable to the slight crossreactivity of the SMP14 clone with cytokeratins.
Development References (2)
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Martin K, Trouche D, Hagemeier C, Sorensen TS, La Thangue NB, Kouzarides T. Stimulation of E2F1/DP1 transcriptional activity by MDM2 oncoprotein. Nature. 1995; 375(6533):691-694. (Clone-specific: Western blot). View Reference
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Picksley SM, Vojtesek B, Sparks A, Lane DP. Immunochemical analysis of the interaction of p53 with MDM2;--fine mapping of the MDM2 binding site on p53 using synthetic peptides. Oncogene. 1994; 9(9):2523-2529. (Immunogen). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.
