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Biotin Mouse Anti-Human CD178
Biotin Mouse Anti-Human CD178

Flow cytometric analysis of human CD178 (FasL) expression on CD178-transfected cells. The mouse T cell lymphoma cell line, L5187Y was untransfected (right panel) or transfected with human CD178 cDNA (left panel), then treated with a metalloproteinase inhibitor, KB8301. KB8301 blocks CD178 cleavage, resulting in high levels of cell surface CD178. Both transfected and untransfected cells were stained with either Biotin Mouse IgG1 κ Isotype Control (Cat. No. 555747; dashed line histogram) or Biotin Mouse Anti-Human CD178 (Cat. No. 556373/556374; solid line histogram) followed by Streptavidin-PE (Cat. No. 554061). Fluorescent histograms were derived from gated events with the side and forward light-scattering characteristics of viable cells.

Flow cytometric analysis of human CD178 (FasL) expression on CD178-transfected cells. The mouse T cell lymphoma cell line, L5187Y was untransfected (right panel) or transfected with human CD178 cDNA (left panel), then treated with a metalloproteinase inhibitor, KB8301. KB8301 blocks CD178 cleavage, resulting in high levels of cell surface CD178. Both transfected and untransfected cells were stained with either Biotin Mouse IgG1 κ Isotype Control (Cat. No. 555747; dashed line histogram) or Biotin Mouse Anti-Human CD178 (Cat. No. 556373/556374; solid line histogram) followed by Streptavidin-PE (Cat. No. 554061). Fluorescent histograms were derived from gated events with the side and forward light-scattering characteristics of viable cells.

Product Details
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BD Pharmingen™
Fas ligand; FASL; FASLG; CD95 ligand; CD95L; CD95-L; TNFSF6; APTL; APT1LG1
Human (QC Testing)
Mouse IgG1
Mouse T lymphoma cells (L5178Y) expressing human FasL
Flow cytometry (Routinely Tested), Western blot (Not Recommended)
20 µl
356
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with biotin under optimum conditions, and unreacted biotin was removed.

Recommended Assay Procedures

Biotin-conjugated NOK-1 (Cat. No. 556376) is recommended for flow cytometry (titrate between 0.06 to 1 µg/1x10^6 cells). Biotin-conjugated NOK-1 is also available in a 100 test size (Cat. No. 556374), and has been optimally titrated for use in flow cytometry. NOK-1 has also been used for immunoprecipitation (see purified format, Cat. No. 556372), and to neutralize the cytotoxic activity of FasL. The NA/LE™ format of NOK-1 (Cat. No. 556371) should be used for all functional assays. NOK-1 and a related human FasL clone, NOK-2 (purified, Cat. No. 556376; BD NA/LE™, Cat. No. 556375) may give different profiles in neutralization assays. It is thought that NOK-1 and NOK-2 recognize different FasL epitopes. Neither NOK-1 and NOK-2 are suggested for western blot analysis. Clone G247-4 (Cat. No. 556387) is suggested for western blot.

Product Notices

  1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
  2. An isotype control should be used at the same concentration as the antibody of interest.
  3. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  4. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  6. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
556374 Rev. 9
Antibody Details
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NOK-1

The NOK-1 monoclonal antibody specifically recognizes CD178. Fas (CD95; APO-1) is a 45 kDa cell surface protein that mediates apoptosis when cross-linked with agonistic anti-Fas antibodies or by Fas ligand (FasL; CD178). Fas belongs to the TNF (Tumor Necrosis Factor)/NGF (Nerve Growth Factor) receptor family, and is expressed in various tissues and cells including the thymus, liver, ovary and lung. CD178 (FasL), a member of the TNF cytokine family, induces apoptosis by binding to Fas, its cell-surface receptor. FasL may exist as either membrane bound or soluble forms and is expressed by activated T and NK cells. FasL may also be constitutively expressed in some immunologically privileged sites, e.g., eye and testis. Fas and FasL play an important role in the induction of apoptosis, and thus regulate a variety of immunological responses.

The NOK-1 antibody clone has been reported to recognize human FasL, recognizing both the membrane bound (FasL) and soluble (sFasL) forms. It is reported that the epitope for NOK-1 has been mapped to the COOH-terminus of FasL, at the region implicated in Fas binding. FasL and sFasL have been reported to migrate at reduced molecular weights of 40 and 26 kDa, respectively. However, the molecular weights observed in a particular sample may vary according to FasL and sFasL glycosylation and breakdown patterns as described in the literature.  The NOK-1 antibody clone is not recommended for the Western blot application.

556374 Rev. 9
Format Details
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Biotin
Biotin is a ubiquitous co-factor (also known as Vitamin B7) that has many properties that make it extremely useful for molecular biology. Biotin has an extremely high affinity for the Avidin family of proteins (Kd = 10-15 M), making it the perfect tool to link two molecules. Biotin labeled antibodies can be combined with any number of Avidin-conjugated probes in order to customize an assay to a particular need. This is especially useful in the case of magnetic cell separation using streptavidin/magnetic bead conjugates, or in the case of flow cytometry using streptavidin/fluorophore conjugates.
Biotin
556374 Rev.9
Citations & References
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Development References (3)

  1. Kayagaki N, Kawasaki A, Ebata T, et al. Metalloproteinase-mediated release of human Fas ligand. J Exp Med. 1995; 182(6):1777-1783. (Immunogen: Cytotoxicity, Flow cytometry). View Reference
  2. Takahashi T, Tanaka M, Brannan CI, et al. Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell. 1994; 76(6):969-976. (Biology). View Reference
  3. Tanaka M, Suda T, Takahashi T, Nagata S. Expression of the functional soluble form of human Fas ligand in activated lymphocytes. EMBO J. 1995; 14(6):1129-1135. (Biology). View Reference
556374 Rev. 9

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For Research Use Only. Not for use in diagnostic or therapeutic procedures.