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/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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Triton is a trademark of the Dow Chemical Company.
Companion Products
The Stat proteins function as both cytoplasmic signal transducers and activators of transcription. The Stat91/84 (the two proteins are the result of alternate splicing-Stat91 has an additional 38 C-terminal amino acids) and Stat113 were the first identified members of this protein family. These three polypeptides contain both SH2 and SH3 domains and have also been described as members of the ISGF3 (interferon-stimulated gene factor 3) complex. With the discovery of additional members of the Stat family (Stats3 & 4), the nomenclature has been revised to indicate the Stat family members in the order of their discovery. Stat 91, 84, and 113 have become Stat1α, Stat1β, and Stat2, respectively. Stat1α is present in a higher concentration than Stat1β in most cell types. In response to IFNα treatment, Stat1α, Stat1β, and Stat2 become tyrosine-phosphorylated and migrate to the nucleus where they join a 48kDa DNA binding protein and subsequently direct the transcription at IFNα responsive elements. In IFN-γ treated cells, Stat1α (but not Stat2) becomes phosphorylated and forms a dimer. It then enters the nucleus and binds to the IFN-γ activated site (GAS) element in order to direct IFN-γ activated transcription.
Development References (5)
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Akira S, Nishio Y, Inoue M, et al. Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway. Cell. 1994; 77(1):63-71. (Biology). View Reference
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Ali MS, Sayeski PP, Bernstein KE. Jak2 acts as both a STAT1 kinase and as a molecular bridge linking STAT1 to the angiotensin II AT1 receptor.. J Biol Chem. 2000; 275(20):15586-93. (Clone-specific: Immunoprecipitation, Western blot). View Reference
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Rayanade RJ, Ndubuisi MI, Etlinger JD, Sehgal PB. Regulation of IL-6 signaling by p53: STAT3- and STAT5-masking in p53-Val135-containing human hepatoma Hep3B cell lines. J Immunol. 1998; 161(1):325-334. (Clone-specific: Immunofluorescence, Western blot). View Reference
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Ruff-Jamison S, Chen K, Cohen S. Induction by EGF and interferon-gamma of tyrosine phosphorylated DNA binding proteins in mouse liver nuclei. Science. 1993; 261(5129):1733-1736. (Biology). View Reference
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Zhou YC, Waxman DJ. Cross-talk between janus kinase-signal transducer and activator of transcription (JAK-STAT) and peroxisome proliferator-activated receptor-alpha (PPARalpha) signaling pathways. Growth hormone inhibition of pparalpha transcriptional activity mediated . J Biol Chem. 1999; 274(5):2672-2681. (Clone-specific: Gel shift, Western blot). 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.
