Human Fc gamma RIIIA/CD16a Antibody

Catalog #: MAB11728 Datasheet / COA / SDS

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MAB11728-100
MAB11728-SP

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Detection of CD16a in Human Spleen via seqIF™ staining on COMET™

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Human Fc gamma RIIIA/CD16a Antibody Summary

Species Reactivity

Human

Specificity

Detects a synthetic peptide specific for human FC gamma R3A around amino acid 55 in Direct ELISA.

Source

Monoclonal Mouse IgG₁ Clone # 1105901

Purification

Protein A or G purified from cell culture supernatant

Immunogen

Synthetic Peptide
Accession # P08637

Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose.

Label

Unconjugated

Applications

Recommended Concentration

Sample

Western Blot

1 µg/mL

Human lung tissue and human spleen tissue

Immunohistochemistry

0.5-15 µg/mL

Immersion fixed paraffin-embedded sections of human liver and spleen

Multiplex Immunofluorescence

5 µg/mL

Immersion fixed paraffin-embedded sections of human spleen

Please Note: Optimal dilutions should be determined by each laboratory for each application. General Protocols are available in the Technical Information section on our website.

Scientific Data

Multiplex Immunofluorescence

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Detection of CD16a in Human Spleen via seqIF™ staining on COMET™ CD16a was detected in immersion fixed paraffin-embedded sections of human Spleen using Mouse Anti-Human CD16a, Monoclonal Antibody (Catalog # MAB11728) at 5ug/mL at 37 ° Celsius for 4 minutes. Before incubation with the primary antibody, tissue underwent an all-in-one dewaxing and antigen retrieval preprocessing using PreTreatment Module (PT Module) and Dewax and HIER Buffer H (pH 9; Epredia Catalog # TA-999-DHBH). Tissue was stained using the Alexa Fluor™ Plus 647 Goat anti-Mouse IgG Secondary Antibody at 1:200 at 37° Celsius for 4 minutes. (Yellow; Lunaphore Catalog # DR647MS) and counterstained with DAPI (blue; Lunaphore Catalog # DR100). Specific staining was localized to the membrane. Protocol available in COMET™ Panel Builder.

Western Blot

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Detection of Human Fc gamma RIIIA/CD16a by Western Blot. Western Blot shows lysates of human lung tissue and human spleen tissue. PVDF membrane was probed with 1 µg/ml of Mouse Anti-Human Fc gamma RIIIA/CD16a Monoclonal Antibody (Catalog # MAB11728) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for Fc gamma RIIIA/CD16a at approximately 50-75 kDa (as indicated). This experiment was conducted under reducing conditions and using Western Blot Buffer Group 1.

Immunohistochemistry

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Detection of Fc gamma RIIIA/CD16a in Human Liver. Fc gamma RIIIA/CD16a was detected in immersion fixed paraffin-embedded sections of human liver using Mouse Anti-Human Fc gamma RIIIA/CD16a Monoclonal Antibody (Catalog # MAB11728) at 1 µg/ml for 1 hour at room temperature followed by incubation with the Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC001) or the HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic (Catalog # VCTS021). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to the cell surface. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Immunohistochemistry

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Detection of Fc gamma RIIIA/CD16a in Human Spleen. Fc gamma RIIIA/CD16a was detected in immersion fixed paraffin-embedded sections of human spleen using Mouse Anti-Human Fc gamma RIIIA/CD16a Monoclonal Antibody (Catalog # MAB11728) at 1 µg/ml for 1 hour at room temperature followed by incubation with the Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC001) or the HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic (Catalog # VCTS021). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to the cell surface. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Reconstitution Calculator

Preparation and Storage

Reconstitution

Reconstitute lyophilized material at 0.2 mg/ml in sterile PBS. For liquid material, refer to CoA for concentration.

Shipping

Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.

Stability & Storage

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.

12 months from date of receipt, -20 to -70 °C as supplied.
1 month, 2 to 8 °C under sterile conditions after reconstitution.
6 months, -20 to -70 °C under sterile conditions after reconstitution.

Background: Fc gamma RIIIA/CD16a

Fc gamma RIIIa is a low/intermediate affinity receptor for polyvalent immune-complexed IgG. It is involved in phagocytosis, secretion of enzymes and inflammatory mediators, antibody-dependent cytotoxicity and clearance of immune complexes (1, 2). In humans, it is a 50-70 kDa type I transmembrane activating receptor expressed by NK cells, T cells, monocytes, and macrophages (1). Fc gamma RIIIb is highly related, sharing 97% amino acid (aa) identity within the extracellular domain (ECD), but is a GPI-linked receptor expressed on human neutrophils and eosinophils (1, 2). The ECD of Fc gamma RIIIa shares 63%, 61%, 65%, 59% and 58% aa identity with mouse Fc gamma RIV, rat Fc gamma RIIIa, feline CD16, bovine CD16 and porcine Fc gamma RIIIb paralogs, respectively. The Fc gamma RIIIa cDNA encodes 254 aa including a 16 aa signal sequence, 191 aa ECD with two C2-type Ig-like domains and five potential N-glycosylation sites, a 22 aa transmembrane (TM) sequence and a 25 aa cytoplasmic domain. In humans, a single nucleotide polymorphism creates high binding (176V) and low binding (176F) forms that, when homozygous, may influence susceptibility to autoimmune diseases or response to therapeutic IgG antibodies (3, 4). Catalog # 4325-FC is expressed as the 176V isoform of Fc gamma RIIIa. Fc gamma RIIIa surface expression requires interaction of an accessory chain, either the common gamma -chain or CD3 zeta (5, 6). Glycosylation patterns, electrophoretic mobility and binding affinity appear to differ between NK cell and monocyte Fc gamma RIIIa (7). The ECD of both Fc gamma RIIIa and b can be proteolytically cleaved and retain binding activity in soluble form (8-11). In monocytes and macrophages, activation and phagocytosis can trigger Fc gamma RIIIa release (11). Soluble Fc gamma RIII can be detected in normal plasma and is increased in rheumatoid arthritis and in coronary artery diseases (9, 10).

References

Nimmerjahn, F. and J.V. Ravetch (2006) Immunity 24:19.
Ravetch, J.V. and B. Perussia (1989) J. Exp. Med. 170:481.
Wu, J. et al. (1997) J. Clin. Invest. 100:1059.
Dall’Ozzo, S. et al. (2004) Cancer Res. 64:4664.
Kim, M.-K. et al. (2003) Blood 101:4479.
Lanier, L.L. et al. (1989) Nature 342:803.
Edberg, J.C. and R.P. Kimberley (1997) J. Immunol. 159:3849.
Li, P. et al. (2007) J. Biol. Chem. 282:6210.
Masuda, M. et al. (2003) J. Rheumatol. 30:1911.
Masuda, M. et al. (2006) Atherosclerosis 188:377.
Webster, N.L. et al. (2006) J. Leukoc. Biol. 79:294.

Long Name

Fc gamma Receptor III A

Entrez Gene IDs

2214 (Human); 304966 (Rat)

Alternate Names

CD16a antigen; CD16a; CD16FCRIIIA; Fc fragment of IgG, low affinity IIIa, receptor (CD16a); Fc fragment of IgG, low affinity IIIa, receptor for (CD16); Fc gamma receptor III-A; Fc gamma RIIIA; FCG3; Fc-gamma receptor III-2 (CD 16); Fc-gamma receptor IIIb (CD16); Fc-gamma RIII; Fc-gamma RIIIa; Fc-gamma RIII-alpha; FCGR3; FCGR3A; FCGRIII; FcgRIIIA; FcR-10; FCRIII; FcRIIIa; IGFR3FcgammaRIIIA; immunoglobulin G Fc receptor III; low affinity III, receptor for (CD16); low affinity immunoglobulin gamma Fc region receptor III-A; neutrophil-specific antigen NA