Upstream / Downstream
Explore pathways related to this product.
CST Antibody Performance Guarantee
To Purchase # 8183S
|8183S||100 µl (50 tests)||$299.00|
|$ 0. 00|
Find answers on our FAQs page.
PTM information and tools available.
Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb (Alexa Fluor® 555 Conjugate) #8183
Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated (+) with IFN-α (right), using Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb (Alexa Fluor® 555 Conjugate) (red) and β-Tubulin (9F3) Rabbit mAb (Alexa Fluor® 488 Conjugate) #3623 (green).Learn more about how we got this image
Gallery: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb (Alexa Fluor® 555 Conjugate) #8183
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
- 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
- Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
- Methanol, 100%
- Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. While stirring, add 30 µl Triton™ X-100.
- Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (9998), mix.
- Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
B. Specimen Preparation - Cultured Cell Lines (IF-IC)
NOTE: Cells should be grown, treated, fixed and stained directly in multiwell plates, chamber slides or on coverslips.
- Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde in 1X PBS.
NOTE: Formaldehyde is toxic, use only in fume hood.
- Allow cells to fix for 15 minutes at room temperature.
- Aspirate fixative, rinse three times in 1X PBS for 5 minutes each.
- Proceed with Immunostaining (Section C).
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
- Methanol Permeabilization Step: Cover cells with ice-cold 100% methanol (use enough to cover completely to a depth of 3–5 mm, DO NOT LET DRY), incubate in methanol for 10 minutes at –20°C, rinse in 1X PBS for 5 minutes.
- Block specimen in Blocking Buffer for 60 minutes.
- While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
- Aspirate blocking solution, apply diluted primary antibody.
- Incubate overnight at 4°C.
- Rinse three times in 1X PBS for 5 minutes each.
- Coverslip slides with Prolong® Gold Antifade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
- For best results examine specimens immediately using appropriate excitation wavelength. For long term storage, store slides flat at 4°C protected from light.
posted November 2006
revised December 2010
protocol id: 220
Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb (Alexa Fluor® 555 Conjugate) detects endogenous levels of Stat1 only when phosphorylated at Tyr701. The antibody detects phosphorylated Tyr701 of both p91 and p84 Stat1. It does not cross-react with the corresponding phospho-tyrosines of other Stat proteins.Species Reactivity: Human, Mouse
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr701 of human Stat1.
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 555 fluorescent dye and tested in-house for immunofluroescent analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb #9167.
The Stat1 transcription factor is activated in response to a large number of ligands (1) and is essential for responsiveness to IFN-α and IFN-γ (2,3). Phosphorylation of Stat1 at Tyr701 induces Stat1 dimerization, nuclear translocation, and DNA binding (4). Stat1 protein exists as a pair of isoforms, Stat1α (91 kDa) and the splice variant Stat1β (84 kDa). In most cells, both isoforms are activated by IFN-α, but only Stat1α is activated by IFN-γ. The inappropriate activation of Stat1 occurs in many tumors (5). In addition to tyrosine phosphorylation, Stat1 is also phosphorylated at Ser727 through a p38 mitogen-activated protein kinase (MAPK)-dependent pathway in response to IFN-α and other cellular stresses (6). Serine phosphorylation may be required for the maximal induction of Stat1-mediated gene activation.
Protein Specific References
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc. The Alexa Fluor® dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc. U.S. Patent No. 5,675,063.