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28764
dsRNA (K1) Mouse mAb
Primary Antibodies
Monoclonal Antibody

dsRNA (K1) Mouse mAb #28764

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  1. IF
Immunofluorescence Image 1: dsRNA (K1) Mouse mAb
Confocal immunofluorescent analysis of Vero cells, mock transfected (left), transfected with poly(I:C) (100 μg/mL, 16 hr; center), or transfected with poly(I:C) and post-processed with ShortCut® RNase III (right), using dsRNA (K1) Mouse mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
To Purchase # 28764S
Cat. # Size Price
28764S
200 µg N/A

Supporting Data

REACTIVITY All
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Mouse IgG2a kappa

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Usage Information

Application Dilution
Immunofluorescence (Immunocytochemistry) 1:800 - 1:3200

Storage

Supplied in a PBS solution that does not contain any stabilizers or preservatives and has been lyophilized. The lyophilized antibody is stable for 24 months when stored at -20°C. Reconstitute using 200 μl of sterile distilled water to create a 1 mg/ml solution. A slight precipitate may be present, but will not interfere with antibody performance. Recommended to centrifuge the reconstituted antibody and use the supernatant. Once reconstituted, this product will freeze at -20°C so it is recommended to aliquot into single-use vials to avoid multiple freeze/thaw cycles.

Protocol

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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) to 9 ml 1X PBS) and 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-Mouse 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 product webpage 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: 148

Specificity / Sensitivity

dsRNA (K1) Mouse mAb recognizes dsRNA where the length of the helix is greater than or equal to 40 base pairs (bp). The ability to recognize dsRNA is independent of the sequence and nucleotide composition of the antigen. The K1 clone shows a higher affinity to poly(I:C) than to other dsRNA antigens (6).

Species Reactivity:

All Species Expected

Source / Purification

Monoclonal antibody is produced by immunizing animals with a nucleic acid sequence.

Background

Double-stranded RNA (dsRNA) is produced during the replication cycle of a broad range of viruses and can trigger the innate immune response. dsRNA is present if cells are infected with a dsRNA virus or it can be generated during the course of single-stranded RNA (ssRNA) virus replication. Host cells do not produce dsRNA so the innate immune response can differentiate between host and viral RNAs by the presence of dsRNA (1). The innate immune response is controlled by several classes of germline-encoded pattern-recognition receptors (PRRs), including retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), Toll-like receptors (TLRs), and NOD-like receptors (NLRs) that sense viral components, such as dsRNA, ssRNA, and DNA (2,3). Activation of PRRs by dsRNA leads to production of type I interferons (IFNs) and proinflammatory cytokines, triggering the adaptive immune response (2,3). Understanding how antiviral responses are initiated, the counter strategies that viruses adopt, and exploring the viral life cycle gives researchers a better comprehension of how to treat infections of different viral diseases (4,5).
  1. Ogden, K.M. and Prasad, B.V. (2015) Oncotarget 6, 28535-6.
  2. Cui, J. et al. (2014) Hum Vaccin Immunother 10, 3270-85.
  3. Takeuchi, O. and Akira, S. (2009) Immunol Rev 227, 75-86.
  4. Knoops, K. et al. (2012) J Virol 86, 2474-87.
  5. Welsch, S. et al. (2009) Cell Host Microbe 5, 365-75.
  6. Schönborn, J. et al. (1991) Nucleic Acids Res 19, 2993-3000.

Limited Uses

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For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DyLight is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.
ProLong is a registered trademark of Life Technologies Corporation.
ShortCut® is a registered trademark of New England Biolabs, Inc.
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