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12869
LGP2 (D3I3L) Rabbit mAb
Primary Antibodies
Monoclonal Antibody

LGP2 (D3I3L) Rabbit mAb #12869

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  1. WB
Western Blotting Image 1 - LGP2 (D3I3L) Rabbit mAb

Western blot analysis of extracts from various cell lines, untreated (-) or treated with Human Interferon-α1 (hIFNα) #8927 (10 ng/ml, overnight; +), using LGP2 (D3I3L) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

Western Blotting Image 2 - LGP2 (D3I3L) Rabbit mAb

Western blot analysis of extracts from THP-1 cells differentiated with TPA #4174 (80 nM, overnight), untreated or LPS-treated (1 μg/ml for indicated times), using LGP2 (D3I3L) Rabbit mAb (upper) or β-Actin (D6A8) #8457 (lower).

Western Blotting Image 3 - LGP2 (D3I3L) Rabbit mAb

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human LGP2 protein (hLGP2-Myc/DDK; +), using LGP2 (D3I3L) Rabbit mAb.

To Purchase # 12869S
Product # Size Price
12869S
100 µl $ 268

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa) 77
Source/Isotype Rabbit IgG

Application Key:

  • W-Western
  • 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
  • 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
Western Blotting 1:1000

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.

Protocol

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Western Blotting Protocol

For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.

A. Solutions and Reagents

From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit

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

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
  3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
  4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
  6. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  7. Nonfat Dry Milk: (#9999).
  8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Bovine Serum Albumin (BSA): (#9998).
  11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
  12. Biotinylated Protein Ladder Detection Pack: (#7727).
  13. Prestained Protein Marker, Broad Range (11-190 kDa): (#13953).
  14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  15. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  16. Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
  5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.
  7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

    NOTE: Loading of prestained molecular weight markers (#13953, 5 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.

  8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
  2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
  3. Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

  1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product webpage) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 ml of TBST.
  3. Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody (#7074 at 1:2000) and anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 ml of TBST.
  5. Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

  1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
  2. Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
  3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised June 2020

Protocol Id: 10

Specificity / Sensitivity

LGP2 (D3I3L) Rabbit mAb recognizes endogenous levels of total LGP2 protein.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val478 of human LGP2 protein.

Background

Antiviral innate immunity depends on the combination of parallel pathways triggered by virus detecting proteins in the Toll-like receptor (TLR) family and RNA helicases, such as Rig-I (retinoic acid-inducible gene I) and MDA-5 (melanoma differentiation-associated antigen 5), which promote the transcription of type I interferons (IFN) and antiviral enzymes (1-3). TLRs and helicase proteins contain sites that recognize the molecular patterns of different virus types, including DNA, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), and glycoproteins. These antiviral proteins are found in different cell compartments; TLRs (i.e. TLR3, TLR7, TLR8, and TLR9) are expressed on endosomal membranes and helicases are localized to the cytoplasm. Rig-I expression is induced by retinoic acid, LPS, IFN, and viral infection (4,5). Both Rig-I and MDA-5 share a DExD/H-box helicase domain that detects viral dsRNA and two amino-terminal caspase recruitment domains (CARD) that are required for triggering downstream signaling (4-7). Rig-I binds both dsRNA and viral ssRNA that contains a 5'-triphosphate end not seen in host RNA (8,9). Though structurally related, Rig-I and MDA-5 detect a distinct set of viruses (10,11). The CARD domain of the helicases, which is sufficient to generate signaling and IFN production, is recruited to the CARD domain of the MAVS/VISA/Cardif/IPS-1 mitochondrial protein, which triggers activation of NF-κB, TBK1/IKKε, and IRF-3/IRF-7 (12-15).

The DExD/H-box family helicase laboratory of genetics and physiology 2 (LGP2, DHX58) is a Rig-I-like receptor (RLR) that lacks the CARD domain and associated signaling ability (6,16). Research studies demonstrate that LGP2 helicase binds dsRNA and inhibits the Rig-I-like receptors Rig-I and MDA-5. Expression of LGP2 is induced by interferon, dsRNA, and viral infection (17). Studies using LGP2-deficient animals demonstrate a complicated interaction between LGP2 and the other RLRs that involves both positive and negative effects on interferon regulation (18-20). In addition, LGP2 may regulate apoptosis, contribute to CD8+ T cell survival, and protect cancer cells from ionizing radiation (21,22).

  1. Yoneyama, M. and Fujita, T. (2007) J Biol Chem 282, 15315-8.
  2. Meylan, E. and Tschopp, J. (2006) Mol Cell 22, 561-9.
  3. Thompson, A.J. and Locarnini, S.A. (2007) Immunol Cell Biol 85, 435-45.
  4. Imaizumi, T. et al. (2002) Biochem Biophys Res Commun 292, 274-9.
  5. Zhang, X. et al. (2000) Microb Pathog 28, 267-78.
  6. Yoneyama, M. et al. (2005) J Immunol 175, 2851-8.
  7. Yoneyama, M. et al. (2004) Nat Immunol 5, 730-7.
  8. Hornung, V. et al. (2006) Science 314, 994-7.
  9. Pichlmair, A. et al. (2006) Science 314, 997-1001.
  10. Kato, H. et al. (2006) Nature 441, 101-5.
  11. Childs, K. et al. (2007) Virology 359, 190-200.
  12. Meylan, E. et al. (2005) Nature 437, 1167-72.
  13. Xu, L.G. et al. (2005) Mol Cell 19, 727-40.
  14. Kawai, T. et al. (2005) Nat Immunol 6, 981-8.
  15. Seth, R.B. et al. (2005) Cell 122, 669-82.
  16. Rothenfusser, S. et al. (2005) J Immunol 175, 5260-8.
  17. Komuro, A. and Horvath, C.M. (2006) J Virol 80, 12332-42.
  18. Venkataraman, T. et al. (2007) J Immunol 178, 6444-55.
  19. Childs, K.S. et al. (2013) PLoS One 8, e64202.
  20. Satoh, T. et al. (2010) Proc Natl Acad Sci U S A 107, 1512-7.
  21. Suthar, M.S. et al. (2012) Immunity 37, 235-48.
  22. Widau, R.C. et al. (2014) Proc Natl Acad Sci U S A, [Epub ahead of print].

Pathways & Proteins

Explore pathways + proteins related to this product.

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