Anti-Glycoprotein 78 Antibody, clone 3F3A

E3 ubiquitin-protein ligase AMFR (UniProt Q9UKV5; also known as AMF receptor, Autocrine motility factor receptor, gp78, RING finger protein 45) is encoded by the RNF45 (also known as AMFR) gene (Gene ID 267) in human. Glycoprotein 78 (gp78) is an endoplasmic reticulum (ER) membrane-anchored E3 ubiquitin ligase that plays a key role in ER-associated degradation (ERAD). Gp78 is localized to a mitochondria-associated ER domain, where it induces mitochondrial fragmentation by targeting the mitochondrial fusion proteins mitofusin 1 and 2 (Mfn1 and Mfn2, respectively) for degradation upon mitochondrial depolarization. Other known ERAD substrates processed by gp78 include ApoB lipoprotein, HMG CoA reductase, CD3-delta, cystic fibrosis transmembrane conductance regulator, the metastasis suppressor KAI1, unglycosylated prion protein (PrP), and the T-cell receptor. Gp78 is a 7-transmembrane protein (a.a. 82-102, 122-142, 145-165, 186-206, 215-235, 276-296, 429-449) with a RING-type Zinc finger (a.a. 341-379), a CUE domain (a.a. 456-498), and a VCP/p97-interacting motif (VIM; a.a. 622-640).

Clone 3F3A detected glycoprotein 78 (gp78) and a ~150 kDa glycoprotein in B16-F1 mouse melanoma cell extract. Cell extract sialidase treatment decreased the antibody′s immunoreactivity toward gp78 and abolished ~150 kDa band detection (Nabi, I.R., et al. (1990). Cancer Res. 50(2):409-414).

Epitope: extracellular domain

Peanut lectin (PNA) resin-purified membrane glycoprotein 78 from B16-F1 cells (Nabi, I.R., and Raz, A. (1987). Int. J. Cancer. 40(3):396-402).

Detect GP78 using this rat monoclonal Anti-Glycoprotein 78, clone 3F3A, Cat. No. MABC949, validated for use in Function Assays, Immunocytochemistry, Immunohistochemistry, and Western Blotting.

Immunocytochemistry: A representative lot detected glycoprotein 78 immunoreactivity colocalized with that of the mitochondria marker OxPhosV in COS-7 cells (Courtesy of I. Robert Nabi, Ph.D., University of British Columbia, Canada).

Affects Function: A representative lot stimulated the mobility of B16-F1 mouse melanoma cells on colloidal gold-coated surface (Nabi, I.R., et al. (1990). Cancer Res. 50(2):409-414).

Immunocytochemistry Analysis: A representative lot detected both surface and cytoplasmic gp78 immunoreactivity by fluorescent immunocytochemistry staining of intact or MeOH/3% paraformaldehyde-fixed and 0.5% Triton X-100-permeablized mouse A31 fibroblasts and A31M angiosarcoma cells (Niinaka, Y., et al. (1998). Cancer Res. 58(12):2667-2674).

Immunocytochemistry Analysis: Clone 3F3A ascites fluid immunolocalized gp78 on the surface of adherent B16-F1 mouse melanoma cells by fluorescent immunocytochemistry staining of 3% paraformaldehyde-fixed cells (Nabi, I.R., et al. (1990). Cancer Res. 50(2):409-414).

Immunohistochemistry Analysis: A representative lot detected an elevated gp78 immunoreactivity in frozen mammary gland sections from 235RLF transgenic mice than age-matched wild type mice (Joshi, B., et al. (2010). J. Biol. Chem. 285(12):8830-8839).

Western Blotting Analysis: A representative lot detected shRNA-mediated gp78 downregulation in HT-1180 human fibrosarcoma cells (Fu, M., et al. (2013). Mol. Biol. Cell. 24(8):1153-1162).

Western Blotting Analysis: A representative lot detected endogenous gp78 and gp78 transgene expression in mammary glands from wild-type and transgenic mice. Clone 3F3A detected a higher gp78 expression in metastatic MDA-435 cells than in non-metastatic MCF7 breast carcinoma cells (Joshi, B., et al. (2010). J. Biol. Chem. 285(12):8830-8839).

Western Blotting Analysis: A representative lot detected much higher gp78 expression in angiosarcoma (human HT-1180 and mouse A31M) than in fibroblast (human IMR90 and mouse A31) lines (Niinaka, Y., et al. (1998). Cancer Res. 58(12):2667-2674).

Western Blotting Analysis: A representative lot detected gp78 and a ~150 kDa band in B16-F1 mouse melanoma cell extract. Autocrine motility factor (AMF) competed against clone 3F3A for binding the gp78 target band. Cell extract sialidase treatment decreased the antibody′s immunoreactivity toward gp78 and abolished ~150 kDa band detection (Nabi, I.R., et al. (1990). Cancer Res. 50(2):409-414).

Research Category
Apoptosis & Cancer

Western Blotting Analysis: A 1:62.5 dilution of this antibody detected glycoprotein 78 (gp78) in 50 µg of HeLa cell lysate.

~78 kDa observed. Target band size appears larger than the calculated molecular weight of 73.00 kDa due to glycosylation. Uncharacterized bands may be observed in some lysate(s).

Format: Purified

Purified rat IgM in buffer containing PBS without azide.

Stable for 1 year at -20°C from date of receipt.
Handling Recommendations: Upon receipt and prior to removing the cap, centrifuge the vial and gently mix the solution. Aliquot into microcentrifuge tubes and store at -20°C. Avoid repeated freeze/thaw cycles, which may damage IgG and affect product performance.

Concentration: Please refer to lot specific datasheet.

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