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62912
Phospho-STING (Ser365) (D1C4T) Rabbit mAb

Phospho-STING (Ser365) (D1C4T) Rabbit mAb #62912

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
M Endogenous Rabbit IgG
IF-IC

Confocal immunofluorescent analysis of Raw 264.7 cells, transfected with poly(dA:dT) (5 ug/ml, 3 hr; left) or mock transfected (right), using Phospho-STING (Ser365) (D1C4T) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our #12727 Immunofluorescence Application Solutions Kit

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 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.
  2. 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.
  3. Blocking Buffer (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 0.5 mL 20X PBS to 9.0 mL dH2O, mix well. While stirring, add 30 µl Triton™ X-100.
  4. 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.
  5. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:

  6. 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 multi-well plates, chamber slides or on coverslips.

  1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.

    NOTE: Formaldehyde is toxic, use only in a fume hood.

  2. Allow cells to fix for 15 min at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
  4. Proceed with Immunostaining (Section C).

C. Immunostaining

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.

  1. Block specimen in Blocking Buffer for 60 min.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr at room temperature in the dark.
  7. Rinse three times in 1X PBS for 5 min each.
  8. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

posted November 2006

revised November 2013

Protocol Id: 24

Application Dilutions
Immunofluorescence (Immunocytochemistry) 1:400
Storage:

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Phospho-STING (Ser365) (D1C4T) Rabbit mAb recognizes endogenous levels of STING protein only when phosphorylated at Ser365.

Species Reactivity:

Mouse

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser365 of mouse STING protein.

Stimulator of interferon genes (STING, TMEM173, MITA) is a transmembrane adaptor protein that is a critical component of the cellular innate immune response to pathogenic cytoplasmic DNA (1,2). STING is a ubiquitously expressed protein found predominantly in the ER (1). The enzyme cGAMP synthase (cGAS) produces the second messenger cyclic-GMP-AMP (cGAMP) in response to cytoplasmic DNA (3,4). cGAMP binds and activates STING (3,4). In addition, detection of cytoplasmic DNA by nucleic acid sensors, including DDX41 or IFI16, results in STING activation (5,6). Following activation, STING translocates with TBK1 to perinuclear endosomes (7). The TBK1 kinase phosphorylates and activates interferon regulatory factors (IRFs) and NF-κB, which leads to the induction of type I interferon and other immune response genes (1,2,7).

Following activation and trafficking, STING gets phosphorylated by ULK1 at Ser366 (Ser365 in mouse), which leads to STING inactivation and eventually lysosomal degradation (8).

  1. Ishikawa, H. and Barber, G.N. (2008) Nature 455, 674-8.
  2. Zhong, B. et al. (2008) Immunity 29, 538-50.
  3. Sun, L. et al. (2013) Science 339, 786-91.
  4. Wu, J. et al. (2013) Science 339, 826-30.
  5. Zhang, Z. et al. (2011) Nat Immunol 12, 959-65.
  6. Unterholzner, L. et al. (2010) Nat Immunol 11, 997-1004.
  7. Ishikawa, H. et al. (2009) Nature 461, 788-92.
  8. Konno, H. et al. (2013) Cell 155, 688-98.
Entrez-Gene Id
72512
Swiss-Prot Acc.
Q3TBT3
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.
DyLight is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.

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Product # Size Price
62912S
100 µl (10 western blots) $ 297.0