Human gp96/HSP90B1 Antibody

    
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  • Species Reactivity
    Human
  • Specificity
    Detects human gp96/HSP90B1 in direct ELISAs and Western blots. Detects mouse and rat gp96/HSP90B1 in Western blots.
  • Source
    Monoclonal Mouse IgG2A Clone # 816803
  • Purification
    Protein A or G purified from hybridoma culture supernatant
  • Immunogen
    E. coli-derived recombinant human gp96/HSP90B1
    Arg503-Arg660
    Accession # P14625
  • Formulation
    Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied as a 0.2 µm filtered solution in PBS.
  • Label
    Unconjugated
Product Datasheets

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Applications
  •  
    Recommended
    Concentration
    Sample
  • Western Blot
    0.2 µg/mL
    See below
  • Simple Western
    2 µg/mL
    See below
  • Immunohistochemistry
    8-25 µg/mL
    See below
  • Immunocytochemistry
    8-25 µg/mL
    See below
  • Knockout Validated
    Gp96/HSP90B1 is specifically detected in HEK293T human embryonic kidney parental cell line but is not detectable in gp96/HSP90B1 knockout HEK293T cell line.
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.
Data Examples
Detection of Human, Mouse, and Rat gp96/HSP90B1 by Western Blot. Western blot shows lysates of 293T human embryonic kidney cell line, HeLa human cervical epithelial carcinoma cell line, A20 mouse B cell lymphoma cell line, CH‑1 mouse B cell lymphoma cell line, and L6 rat myoblast cell line. PVDF membrane was probed with 0.2 µg/mL of Mouse Anti-Human gp96/HSP90B1 Monoclonal Antibody (Catalog # MAB7606) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for gp96/HSP90B1 at approximately 100 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
Immunocytochemistry
gp96/HSP90B1 in HeLa Human Cell Line. gp96/HSP90B1 was detected in immersion fixed HeLa human cervical epithelial carcinoma cell line using Mouse Anti-Human gp96/HSP90B1 Monoclonal Antibody (Catalog # MAB7606) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.
Immunohistochemistry
gp96/HSP90B1 in Human Mesothelioma. gp96/HSP90B1 was detected in immersion fixed paraffin-embedded sections of human mesothelioma using Mouse Anti-Human gp96/HSP90B1 Monoclonal Antibody (Catalog # MAB7606) at 15 µg/mL overnight at 4 °C. Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). Tissue was stained using the Anti-Mouse HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS002) and counterstained with hematoxylin (blue). Specific staining was localized to plasma membranes and cytoplasm. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.
Detection of Human gp96/HSP90B1 by Simple WesternTM. Simple Western lane view shows lysates of HeLa human cervical epithelial carcinoma cell line, loaded at 0.5 mg/mL. A specific band was detected for gp96/HSP90B1 at approximately 108 kDa (as indicated) using 2 µg/mL of Mouse Anti-Human gp96/HSP90B1 Monoclonal Antibody (Catalog # MAB7606). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.
Knockout Validated
Western Blot Shows Human gp96/HSP90B1 Specificity by Using Knockout Cell Line. Western blot shows lysates of HEK293T human embryonic kidney parental cell line and gp96/HSP90B1 knockout HEK293T cell line (KO). PVDF membrane was probed with 0.2 µg/mL of Mouse Anti-Human gp96/HSP90B1 Monoclonal Antibody (Catalog # MAB7606) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for gp96/HSP90B1 at approximately 100 kDa (as indicated) in the parental HEK293T cell line, but is not detectable in knockout HEK293T cell line. GAPDH (Catalog # MAB5718) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
Preparation and Storage
  • Reconstitution
    Sterile PBS to a final concentration of 0.5 mg/mL.
    Reconstitution Buffer Available
  • Shipping
    The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below. *Small pack size (SP) is shipped with polar packs. Upon receipt, store it immediately at -20 to -70 °C
  • 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: gp96/HSP90B1
Glycoprotein 96 (gp96; also endoplasmin, GRP-94, TRA1 and HSP90B1) is a 94-100 kDa member of the HSP 90 family of proteins. gp96 is a ubiquitously-expressed ER resident protein that is found in a preformed complex with BiP, CaBP1 and UDP-glucosyltransferase. This is a chaperone complex that binds unfolded protein substrates. When folded properly, the substrate is forwarded to calnexin-containing chaperone complexes that promote its maturation. gp96 clients are restricted and include disulfide-bonded integrins, TLRs, LDLR and CD180. Within the complex, gp96 exists as a disulfide-linked homodimer that may form higher-order oligomers. gp96 also appears on the cell surface and may serve as a receptor for bacteria. Mature human gp96 is a 782 amino acid (aa) membrane-associated protein
(aa 22-803). It is not a transmembrane protein but utilizes an ER retention signal (aa 800-803) to interact with the ER membrane. The molecule possesses a
HATPase-C like region (aa 98-219) plus multiple ATP binding and two utilized phosphorylation sites. Over aa 503-660, human gp96 shares 98% aa sequence identity with mouse gp96.
  • Long Name:
    Heat Shock Protein 90 beta 1
  • Entrez Gene IDs:
    7184 (Human); 22027 (Mouse); 362862 (Rat)
  • Alternate Names:
    94 kDa glucose-regulated protein; ECGP; Endoplasmin; endothelial cell (HBMEC) glycoprotein; glucose regulated protein, 94 kDa; gp96; GP96ECGP; Grp94; GRP-94; GRP94gp96 homolog; Heat shock protein 90 kDa beta member 1; heat shock protein 90kDa beta (Grp94), member 1; HSP90B1; TRA1endoplasmin; tumor rejection antigen (gp96) 1; Tumor rejection antigen 1; Tumor rejection antigen-1 (gp96)
Related Research Areas

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