Fas/TNFRSF6/CD95 Antibody (R-125224) - Chimeric - Azide and BSA Free

Novus Biologicals, part of Bio-Techne | Catalog # NBP2-81113

Recombinant Monoclonal Antibody
Novus Biologicals, part of Bio-Techne

Key Product Details

Species Reactivity

Human

Applications

ELISA, Flow Cytometry, Functional, Immunocytochemistry/ Immunofluorescence, Western Blot

Label

Unconjugated

Antibody Source

Recombinant Monoclonal Rabbit IgG Kappa Clone # R-125224

Format

Azide and BSA Free

Concentration

1 mg/ml

View all Fas/TNFRSF6/CD95 Antibodies »

Product Specifications

Immunogen

R-125224 is generated by the humanization of the murine HFE7A anti-Fas/TNFRSF6/CD95 antibody by grafting the CDR regions to the framework regions of the human 8E10 antibody and substituting key framework residues from the murine antibody into the 8E10 sequence. The original HFE7A was derived from a hybridoma cell line generated by the fusion of NS1 myeloma cells with splenocytes from Fas-deficient mice which had been immunized with partially purified recombinant human Fas-AIC2A chimera protein consisting of the extracellular region of human Fas/TNFRSF6/CD95 antigen (aa -16 to 150) and the extracellular region of the murine IL-3 receptor AIC2 (aa 3-423). The HFE7A hybridoma was selected after screening by flow cytometry for the production of antibodies with the ability to bind to the WR19L12a transformed murine T cell lymphoma cell line expressing human Fas/TNFRSF6/CD95 or the L5178YA1 cell line expressing murine Fas/TNFRSF6/CD95, but not to the parental WR19L or L5178Y cells.

Specificity

R-125224 binds to the extracellular portion of human Fas/TNFRSF6/CD95 at an eptiope consisting of the sequence RTQNTKCRCK (aa 105-114) (pmid: 11754745). Fas is a type I membrane protein which belongs to the tumor necrosis factor (TNF) receptor/nerve growth factor (NGF) receptor superfamily. It is able to transduce apoptotic signals into the cell when bound by its ligand FasL (Fas ligand), which is primarily expressed in activated T lymphoid-myeloid lineage cells, in the eye, in reproductive organs and in some tumors. The Fas-FasL system is known to play an important role in maintaining the immune system as mice with Fas-defective lymphoproliferation (lpr) and FasL-defective generalized lymphoproliferative disease (gld) mutations develop massive lymphadenopathy and autoimmune diseases.

Clonality

Monoclonal

Host

Rabbit

Isotype

IgG Kappa

Scientific Data Images for Fas/TNFRSF6/CD95 Antibody (R-125224) - Chimeric - Azide and BSA Free

Western Blot: Fas/TNFRSF6/CD95 Antibody (R-125224)ChimericAzide and BSA Free [NBP2-81113]

Western Blot: Fas/TNFRSF6/CD95 Antibody (R-125224)ChimericAzide and BSA Free [NBP2-81113]

Western Blot: Fas/TNFRSF6/CD95 Antibody (R-125224) - Chimeric [NBP2-81113] - Western Blot using Fas/TNFRSF6/CD95 Antibody (R-125224) [NBP2-81113]. Human testis (A) and human ovary (B) lysate samples (35ug protein in RIPA buffer) were resolved on a 10% SDS PAGE gel and blots probed with the chimeric rabbit IgG version of R-125224 [NBP2-81113] at 2 ug/ml before detection using an anti-rabbit secondary antibody. A primary incubation of 1h was used and protein was detected by chemiluminescence. The expected running size for unmodified Fas is 37.7kDa, but this protein is glycosylated at several positions leading to the observed running size.
Immunocytochemistry/ Immunofluorescence: Fas/TNFRSF6/CD95 Antibody (R-125224) - Chimeric - Azide and BSA Free [NBP2-81113]

Immunocytochemistry/ Immunofluorescence: Fas/TNFRSF6/CD95 Antibody (R-125224) - Chimeric - Azide and BSA Free [NBP2-81113]

Immunocytochemistry/Immunofluorescence: Fas/TNFRSF6/CD95 Antibody (R-125224) - Chimeric [NBP2-81113] - Immunofluorescence staining of fixed MCF7 cells with [NBP2-81113]. Immunofluorescence analysis of paraformaldehyde fixed MCF7 cells permeabilized with 0.15% Triton and stained with the chimeric mouse IgG1 version of R-125224 (NBP2-81113) at 10 ug/ml for 1h followed by Alexa Fluor 488 secondary antibody (2 ug/ml), showing membrane staining. The nuclear stain is DAPI (blue). Panels show from left-right, top-bottom NBP2-81113, DAPI, merged channels and an isotype control. The isotype control was stained with an anti-unknown specificity antibody followed by Alexa Fluor 488 secondary antibody.
Flow Cytometry: Fas/TNFRSF6/CD95 Antibody (R-125224) - Chimeric - Azide and BSA Free [NBP2-81113]

Flow Cytometry: Fas/TNFRSF6/CD95 Antibody (R-125224) - Chimeric - Azide and BSA Free [NBP2-81113]

Flow Cytometry: Fas/TNFRSF6/CD95 Antibody (R-125224) - Chimeric [NBP2-81113] - Flow-cytometry using the Fas/TNFRSF6/CD95 Antibody (R-125224) [NBP2-81113]. Jurkat cells were fixed using 2% PFA, permeabilised using 0.5% Triton and stained with unimmunized rabbit IgG antibody (MOPC-21; isotype control, black line) or the rabbit IgG-chimeric version of R-125224 (NBP2-81113, blue line) at a dilution of 1:100 for 1h at RT. After washing, bound antibody was detected using a goat anti-rabbit IgG AlexaFluor 488 antibody at a dilution of 1:1000 and cells analyzed using a FACSCanto flow-cytometer.

Applications for Fas/TNFRSF6/CD95 Antibody (R-125224) - Chimeric - Azide and BSA Free

Application
Recommended Usage

ELISA

Optimal dilutions of this antibody should be experimentally determined.

Flow Cytometry

Optimal dilutions of this antibody should be experimentally determined.

Functional

Optimal dilutions of this antibody should be experimentally determined.

Immunocytochemistry/ Immunofluorescence

Optimal dilutions of this antibody should be experimentally determined.

Western Blot

Optimal dilutions of this antibody should be experimentally determined.
Application Notes
This chimeric rabbit antibody was made using the variable domain sequences of the original Human IgG1 format, for improved compatibility with existing reagents, assays and techniques. R-125224 shows the same binding affinity and the same ability to induce apoptosis in WR19L12a cells that express human Fas as the parental murine HFE7A antibody. R-125224 selectively induces apoptosis in type I activated lymphocytes but not in type II cells. R-125224 is able to induce apoptosis in the human lymphoid cell lines H9 and SKW6.4, as well as activated human lymphocytes, when cross-linked with anti-hIgG secondary antibodies. The antibody is unable to induce apoptosis in HPB-ALL cells, Jurkat cells or human hepatocytes. R-125224 has been used in vivo where it has been shown to greatly reduce the number of activated human human CD3+ Fas+ T cells in a SCID mouse model possessing a functional human immune system. Fas antigen tissue distribution in cynomolgus monkeys with collagen-induced arthritis at the arm joint (CIA monkeys) has been studied using [125I]-Labeled R-125224. High radioactivity in the bone marrow, thymus, lungs, liver, adrenals, spleen, ovaries, axillary lymph node and mesenteric lymph node compared to the radioactivity in the plasma was observed, which correlates with Fas expression. Fas can also be detected by R-125224 by ELISA.

Formulation, Preparation, and Storage

Purification

Protein A purified

Formulation

PBS

Format

Azide and BSA Free

Preservative

0.02% Proclin 300

Concentration

1 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C for up to 3 months. For longer storage, aliquot and store at -20C.

Background: Fas/TNFRSF6/CD95

Tumor Necrosis Family Receptor (TNFR) superfamily member Fas, also known as CD95, APO-1, and TNFRSF6, is a 40-50 kDa type I transmembrane glycoprotein that is traditionally considered a death receptor but also functions in non-apoptotic signaling (1-4). The human Fas/TNFRSF6/CD95 protein is encoded by the FAS gene which contains 9 exons and is located on chromosome 10 (10q23.3-4) (1,2). The mature canonical Fas/TNFRSF6 protein isoform is 335 aa in length, which includes the signal sequence, and has a theoretical molecular weight of 37.7 kDa (1,5). The protein contains an extracellular domain (ECD) consisting of three calcium rich domains (CRDs), a transmembrane domain (TM), and an intracellular domain (ICD) comprised of a calcium-inducing domain (CID) and characteristic dead domain (DD) (1,2,5,6). The Fas protein is expressed on the plasma membrane of activated lymphocytes as a homotrimer formed via CRD1 interactions (1,2,3,6). The DD is crucial for apoptotic signaling which is triggered by the Fas receptor binding its ligand, Fas ligand (FasL) (1,2,6,7). Upon Fas-FasL interaction, the DD recruits an adapter protein Fas-associated DD (FADD) and procaspase-8, generating the death-inducing signaling complex (DISC) (1-4,6-8). Formation of DISC activates caspase-8 and leads to cleavage of caspase-3, initiating a caspase-signaling cascade and cell death (1-4,6-8).

Fas-FasL-mediated apoptosis is important in immune homeostasis and removal of autoreactive T cells, autoreactive B cells, cytotoxic natural killer (NK) cells, and more (1,2,7). Dysfunction and mutations in the Fas receptor and the Fas-FasL signaling axis is associated a loss of apoptotic signaling and removal of autoreactive cells, which correlates with several autoimmune diseases including systemic lupus erythematosus (SLE), autoimmune lymphoproliferative syndrome (ALPS), and multiple sclerosis (MS) (1-4,6,7). In addition to apoptosis and cell death signaling, FasL/TNFRSF6/CD95 mediates other pathways involved in proliferation, survival, and differentiation (3,4,6,8). More specifically, Fas has been shown to activate the NF-kappaB pathway, driving innate immunity which includes IL-1beta production and functioning in host defense (3,4,6,8). Fas is also involved in adaptive immunity playing a role in co-stimulation of CD4+ and CD8+ T cell activation as well as precocious differentiation of naive cells to effector memory T cells (3,4,6). Differentiation into effector memory T cells shows protection against autoimmunity but also limits antitumor response to a form of cancer immunotherapy called adoptive cell transfer (ACT) (3,4). The non-apoptotic roles of the Fas/TNFRSF6/CD95 receptor highlight its potential as a target for both treating autoimmune diseases and in cancer immunotherapy (3,4).

References

1. Singh R, Pradhan V, Patwardhan M, Ghosh K. APO-1/Fas gene: Structural and functional characteristics in systemic lupus erythematosus and other autoimmune diseases. Indian J Hum Genet. 2009;15(3):98-102. https://doi.org/10.4103/0971-6866.60184

2. Magerus A, Bercher-Brayer C, Rieux-Laucat F. The genetic landscape of the FAS pathway deficiencies. Biomed J. 2021;44(4):388-399. https://doi.org/1010.1016/j.bj.2021.06.005

3. Guegan JP, Legembre P. Nonapoptotic functions of Fas/CD95 in the immune response. FEBS J. 2018;285(5):809-827. https://doi.org/10.1111/febs.14292

4. Yi F, Frazzette N, Cruz AC, Klebanoff CA, Siegel RM. Beyond Cell Death: New Functions for TNF Family Cytokines in Autoimmunity and Tumor Immunotherapy. Trends Mol Med. 2018;24(7):642-653. https://doi.org/10.1016/j.molmed.2018.05.004

5. Uniprot (P25445)

6. Guegan JP, Ginestier C, Charafe-Jauffret E, et al. CD95/Fas and metastatic disease: What does not kill you makes you stronger. Semin Cancer Biol. 2020;60:121-131. https://doi.org/10.1016/j.semcancer.2019.06.004

7. Volpe E, Sambucci M, Battistini L, Borsellino G. Fas-Fas Ligand: Checkpoint of T Cell Functions in Multiple Sclerosis. Front Immunol. 2016;7:382. Published 2016 Sep 27. https://doi.org/10.3389/fimmu.2016.00382

8. Cullen SP, Martin SJ. Fas and TRAIL 'death receptors' as initiators of inflammation: Implications for cancer. Semin Cell Dev Biol. 2015;39:26-34. https://doi.org/10.1016/j.semcdb.2015.01.012

Long Name

Fibroblast-associated

Alternate Names

Apo-1, APT1, CD95, TNFRSF6

Gene Symbol

FAS

Additional Fas/TNFRSF6/CD95 Products

Product Documents for Fas/TNFRSF6/CD95 Antibody (R-125224) - Chimeric - Azide and BSA Free

Product Datasheets

Certificate of Analysis

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Product Specific Notices for Fas/TNFRSF6/CD95 Antibody (R-125224) - Chimeric - Azide and BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

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