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31469
Ly-6G/Ly-6C (Gr-1) (RB6-8C5) Rat mAb
Primary Antibodies

Ly-6G/Ly-6C (Gr-1) (RB6-8C5) Rat mAb #31469

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  1. IF

Confocal immunofluorescent analysis of mouse spleen using Ly-6G/Ly-6C (Gr-1) (RB6-8C5) Rat mAb (green) and CD11c (D1V9Y) Rabbit mAb #97585 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

To Purchase # 31469S
Product # Size Price
31469S
100 µl $ 268

Supporting Data

REACTIVITY M
SENSITIVITY Endogenous
MW (kDa)
Isotype Rat IgG2b

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
Immunofluorescence (Frozen) 1:50 - 1:200

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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Immunofluorescence (Frozen)

A. Solutions and Reagents

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 9.5 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-Rat secondary antibodies:

  6. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Frozen/Cryostat Sections (IF-F)

  1. For fixed frozen tissue proceed with Immunostaining (Section C).
  2. For fresh, unfixed frozen tissue, please fix immediately, as follows:
    1. Cover sections with 4% formaldehyde dilute in 1X PBS.

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

    2. Allow sections to fix for 15 minutes at room temperature.
    3. Aspirate liquid, rinse three times in 1X PBS for 5 minutes 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 July 2016

Protocol Id: 152

Specificity / Sensitivity

Ly-6G/Ly-6C (Gr-1) (RB6-8C5) Rat mAb recognizes endogenous levels of total Ly-6G and Ly-6C proteins. This antibody detects an epitope within the extracellular domain.

Species Reactivity:

Mouse

Source / Purification

This monoclonal antibody was purified from tissue culture supernatant via affinity chromatography.

Background

The Ly-6 complex is a series of genes found on chromosome 15. These genes code for a number of different proteins that can be used as surface markers. The family members vary in their biologic expression and have been shown to be involved in cell signaling and cell adhesion (1). The structure of these proteins includes a motif known as the LU domain that has three loops comprised of disulfide bonds. These bonds are formed by 8 to 10 cysteines that can cause differences in the length of the loops as well as the sequences at each tip (2,4). There are 11 known Ly-6 genes on murine chromosome 15 that code for different proteins. These family members, excluding secreted Ly6/Plaur domain containing 1 coded by the Slurp1 gene, are attached to the cell surface by a GPI anchor near the C terminus. The structure of these proteins may play a role in transmembrane interactions, and downstream signaling cascades (1,2).

Ly-6 proteins have been widely used as differentiation markers on hematopoietic cells. The ability to isolate and express specific Ly-6 antibodies through hybridoma technology has allowed researchers to identify unique proteins (1). These proteins are expressed on subsets of immune cells at different stages of development, such as T cells, B cells, monocytes, granulocytes, and macrophages (1-5). 

  1. Bamezai, A. Arch Immunol Ther Exp (Warsz) 52, 255-66.
  2. Lee, P.Y. et al. (2013) J Leukoc Biol 94, 585-94.
  3. Fleming, T.J. et al. (1993) J Immunol 151, 2399-408.
  4. Tsetlin, V. (1999) Eur J Biochem 264, 281-6.
  5. Pflugh, D.L. et al. (2000) J Immunol 165, 313-21.

Pathways & Proteins

Explore pathways + proteins related to this product.

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

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