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76651
dsRNA (J2) Mouse mAb
Primary Antibodies
Monoclonal Antibody

dsRNA (J2) Mouse mAb #76651

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  1. IF
Immunofluorescence Image 1: dsRNA (J2) Mouse mAb
Immunofluorescent analysis of Vero cells, mock-infected (left) versus SARS-CoV-2-infected (right; 24 hr), using dsRNA (J2) Mouse mAb (green) and DAPI #4083 (blue). Data was kindly provided by Autumn Grimins and John Connor, Boston University.
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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
  • C&R-CUT&RUN
  • C&T-CUT&Tag
  • DB-Dot Blot
  • eCLIP-eCLIP
  • IF-Immunofluorescence
  • F-Flow Cytometry

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
  • Rab-Rabbit
  • All-All Species Expected

Product Usage Information

Application Dilution
Immunofluorescence (Immunocytochemistry) 1:500 - 1:1000

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), Modified

NOTE: Some CST antibodies work optimally using an alternate protocol. Please see the protocol on the product web page for product-specific recommendations.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1L 1X PBS, add 100 ml 10X Wash Buffer, Phosphate Buffered Saline (#12528) to 900 ml dH2O, mix. Adjust pH to 8.0.
  2. 4% Formaldehyde, Methanol-Free: (#47746): Use fresh.
  3. Permeabilization Buffer: (1X PBS / 0.5% Triton X-100 detergent) To prepare 10 ml, add 50 ul Triton X-100 detergent to 10 ml 1X PBS and mix well.
  4. Blocking Buffer: (1X PBS / 3% BSA): To prepare 10 ml, add 0.3 g BSA (#9998) to 10 mL 1X PBS, mix well. Store at 4°C.
  5. Fluorochrome-conjugated Secondary Antibody: Use a secondary antibody that is reactive to your primary antibody host species (e.g., mouse). Click here for a list of secondary antibodies approved for immunofluorescence.

B. Fixation and Permeabilization

NOTE: All subsequent incubations should be carried out at room temperature (20-25°C) unless noted otherwise.

  1. Remove culture media from cells.
  2. Rinse once with PBS.
  3. Add 4% Formaldehyde and incubate at room temperature for 15 minutes on rocker.
  4. Remove formaldehyde.
  5. Wash twice with PBS for 5 minutes each.
  6. Incubate cells with Permeabilization Buffer at room temperature for 45 minutes on rocker.
  7. Wash twice with PBS for 5 minutes each.

C. Immunostaining

  1. Incubate cells with Blocking Buffer at room temperature for 45 minutes on rocker.
  2. Wash once with PBS for 5 minutes.
  3. While blocking, prepare primary antibody by diluting in PBS (see product website for recommended dilution range).
  4. Aspirate blocking solution then apply diluted primary antibody.
  5. Incubate overnight at 4°C in a humidity chamber.
  6. Wash twice with PBS for 5 minutes each.
  7. Incubate cells in fluorochrome-conjugated secondary antibody diluted in PBS at room temperature for 1 hour. Do not allow cells to dry and protect from light.
  8. Wash twice with PBS for 5 minutes each.
  9. Counterstain as appropriate.

    NOTE: When including fluorescent cellular dyes in your experiment (DNA dyes, etc.), please refer to the dye product page for its recommended protocol. View our listing of cellular dyes validated for use in immunofluorescence.

  10. Mount samples for imaging.
  11. For long-term storage, store samples at 4°C protected from light.

Protocol Id: 2685

Specificity / Sensitivity

dsRNA (J2) 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 J2 clone shows a higher affinity to natural dsRNA 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

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
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