Clusterin/APOJ Antibody (350227) [Unconjugated]

Western blot shows lysates of human liver tissue and human serum. PVDF membrane was probed with 0.5 µg/mL of Mouse Anti-Human Clusterin Monoclonal Antibody (Catalog # MAB2937) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). Specific bands were detected for Clusterin Precursor at approximately 60-65 kDa and Clusterin a chain at approximately 36 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Clusterin was detected in immersion fixed frozen sections of mouse spleen using Mouse Anti-Human Clusterin Monoclonal Antibody (Catalog # MAB2937) at 5 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC001). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Clusterin was detected in immersion fixed paraffin-embedded sections of human prostate cancer tissue using Mouse Anti-Human Clusterin Monoclonal Antibody (Catalog # MAB2937) at 5 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC001). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Identification of a mitochondrial CLU protein isoform.(A) DIV 9 Mitotracker-stained (green) primary neurons were probed for CLU immunoreactivity using anti-CLU H-330 (red) and visualized using 40X confocal microscopy. (B) Pure cortical mitochondria were isolated as indicated. Equal concentrations of whole tissue lysate, crude mitochondria, and pure mitochondria were analyzed via SDS-PAGE and probed for CLU immunoreactivity using anti-CLU H-330 (left panel) and anti-CLU M-18 (right panel) (n = 3 independent isolations). Biochemical characterization of isolated fractions was performed using a panel of organelle-specific antibodies: voltage-dependent anion channels (VDAC) (mitochondria), calnexin and calreticulin (ER), and Gapdh (cytosol). (C) Crude and pure mitochondria were isolated and subjected to endoglycosidase treatment using PNGase F and Endo H. Deglycosylated mitochondrial lysates were then analyzed for CLU immunoreactivity using anti-CLU M-18. Red font: deglycosylated protein isoforms; blue font: isoforms that were unaffected by glycosidase treatment; green asterisk: excess Endo H enzyme.Positive controls for deglycosylation studies.RNase B, a high mannose glycoprotein, has a single N-linked glycosylation site and was used as a positive control for endoglycosidases that cleave N-linked carbohydrates. Fetuin, a glycoprotein containing sialylated N-linked and O-linked glycans, was used as a positive control for endoglycosidases that cleave both N-linked and O-linked carbohydrates. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/31738162), licensed under a CC-BY license. Not internally tested by R&D Systems.

Identification of a mitochondrial CLU protein isoform.(A) DIV 9 Mitotracker-stained (green) primary neurons were probed for CLU immunoreactivity using anti-CLU H-330 (red) and visualized using 40X confocal microscopy. (B) Pure cortical mitochondria were isolated as indicated. Equal concentrations of whole tissue lysate, crude mitochondria, and pure mitochondria were analyzed via SDS-PAGE and probed for CLU immunoreactivity using anti-CLU H-330 (left panel) and anti-CLU M-18 (right panel) (n = 3 independent isolations). Biochemical characterization of isolated fractions was performed using a panel of organelle-specific antibodies: voltage-dependent anion channels (VDAC) (mitochondria), calnexin and calreticulin (ER), and Gapdh (cytosol). (C) Crude and pure mitochondria were isolated and subjected to endoglycosidase treatment using PNGase F and Endo H. Deglycosylated mitochondrial lysates were then analyzed for CLU immunoreactivity using anti-CLU M-18. Red font: deglycosylated protein isoforms; blue font: isoforms that were unaffected by glycosidase treatment; green asterisk: excess Endo H enzyme.Positive controls for deglycosylation studies.RNase B, a high mannose glycoprotein, has a single N-linked glycosylation site and was used as a positive control for endoglycosidases that cleave N-linked carbohydrates. Fetuin, a glycoprotein containing sialylated N-linked and O-linked glycans, was used as a positive control for endoglycosidases that cleave both N-linked and O-linked carbohydrates. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/31738162), licensed under a CC-BY license. Not internally tested by R&D Systems.

• Reactivity: Hu
• Applications: WB, IHC, IP
• Clone: 350227
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: Unconjugated
• Concentration: LYOPH

Clusterin/APOJ Antibody (350227) [Unconjugated] Summary

• Immunogen: Mouse myeloma cell line NS0-derived recombinant human Clusterin isoform 1
  Asp23-Arg227 (beta) & Ser228-Glu449 (alpha)
  Accession # NP_001822
• Specificity: Detects human Clusterin in direct ELISAs and Western blots. Does not cross-react with recombinant human Clusterin-like 1, recombinant mouse Clusterin, or recombinant rat Clusterin.
• Source: N/A
• Isotype: IgG2b
• Clonality: Monoclonal
• Host: Mouse
• Gene: CLU
• Purity Statement: Protein A or G purified from hybridoma culture supernatant

Applications/Dilutions

• Dilutions:
  • Immunohistochemistry 5-25 ug/mL
  • Immunoprecipitation 25 ug/mL
  • Western Blot 2 ug/mL

Packaging, Storage & Formulations

• 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.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.
• Preservative: No Preservative
• Concentration: LYOPH
• Reconstitution Instructions: Reconstitute at 0.5 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Clusterin/APOJ Antibody (350227) [Unconjugated]

• 40
• 40, sulfated glycoprotein 2
• Aging-associated gene 4 protein
• aging-associated protein 4
• APOJ
• apo-J
• Apolipoprotein J
• CLI
• CLIclusterin (complement lysis inhibitor, SP-40
• CLU
• Clusterin
• Complement cytolysis inhibitor
• complement lysis inhibitor
• Complement-associated protein SP-40
• Ku70-binding protein 1
• KUB1SGP2
• MGC24903
• NA1/NA2
• SGP-2
• SP-40
• sulfated glycoprotein 2
• Testosterone-repressed prostate message 2
• testosterone-repressed prostate message 2, apolipoprotein J)
• TRPM-2
• TRPM-2TRPM2

Background

Clusterin, also known as Apolipoprotein J, Sulfated Glycoprotein 2 (SGP-2), TRPM-2, and SP-40, is a secreted multi-functional protein that was named for its ability to induce cellular clustering. It binds a wide range of molecules and may function as a chaperone of misfolded extracellular proteins. It also participates in the control of cell proliferation, apoptosis, and carcinogenesis (1, 2). Clusterin is predominantly expressed in adult testis, ovary, adrenal gland, liver, heart, and brain and in many epithelial tissues during embryonic development (3). Human Clusterin is synthesized as a precursor that contains two coiled coil domains, three nuclear localization signals (NLS), and one heparin binding domain (4-6). Intracellular cleavages of the precursor remove the signal peptide and generate comparably sized alpha and beta chains which are secreted as an 80 kDa N-glycosylated disulfide-linked heterodimer (7, 8). Mature human Clusterin shares 77% amino acid sequence identity with mouse and rat Clusterin. High μg/mL concentrations of Clusterin circulate predominantly as a component of high density lipoprotein particles, and these are internalized and degraded through interactions with LRP-2/Megalin (9, 10). In human, an alternately spliced 50 kDa isoform of Clusterin (nCLU) lacks the signal peptide and remains intracellular (5, 11). This molecule is neither glycosylated nor cleaved into alpha and beta chains (11). In the cytoplasm, nCLU destabilizes the actin cytoskeleton and inhibits NF kappa B activation (12, 13). Cellular exposure to ionizing radiation promotes the translocation of nCLU to the nucleus where it interacts with Ku70 and promotes apoptosis (5, 11). This function contrasts with the cytoprotective effect of secreted Clusterin (14). During colon cancer tumor progression there is a downregulation of the intracellular form and an upregulation of the glycosylated secreted form (11).

1. Carver, J.A. et al. (2003) IUBMB Life 55:661.
2. Shannan, B. et al. (2006) Cell Death Differ. 13:12.
3. French, L.E. et al. (1993) J. Cell Biol. 122:1119.
4. Kirszbaum, L. et al. (1989) EMBO J. 8:711.
5. Leskov, K.S. et al. (2003) J. Biol. Chem. 278:11590.
6. Pankhurst, G.J. et al. (1998) Biochemistry 37:4823.
7. Burkey, B.F. et al. (1991) J. Lipid. Res. 32:1039.
8. de Silva, H.V. et al. (1990) J. Biol. Chem. 265:14292.
9. Jenne, D.E. et al. (1991) J. Biol. Chem. 266:11030.
10. Kounnas, M.Z. et al. (1995) J. Biol. Chem. 270:13070.
11. Pucci, S. et al. (2004) Oncogene 23:2298.
12. Moretti, R. M. et al. (2007) Cancer Res. 67:10325.
13. Santilli, G. et al. (2003) J. Biol. Chem. 278:38214.
14. Trougakos, I.P. et al. (2004) Cancer Res. 64:1834.

Limitations

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.

Publications for Clusterin/APOJ Antibody (MAB2937)(6)

Publications using MAB2937

• Li J, Jaiswal MK, Chien JF et al. Divergent single cell transcriptome and epigenome alterations in ALS and FTD patients with C9orf72 mutation Nat Commun 9/15/2023 [PMID: 37714849]
• Li J, Jaiswal MK, Chien JF et al. Divergent single cell transcriptome and epigenome alterations in ALS and FTD patients with C9orf72 mutation Nat Commun 2023-09-15 [PMID: 37714849]
• K Ceyzériat, T Zilli, P Millet, N Koutsouvel, G Dipasquale, C Fossey, T Cailly, F Fabis, GB Frisoni, V Garibotto, BB Tournier Low-dose brain irradiation normalizes TSPO and CLUSTERIN levels and promotes the non-amyloidogenic pathway in pre-symptomatic TgF344-AD rats Journal of Neuroinflammation, 2022-12-22;19(1):311. 2022-12-22 [PMID: 36550510] (Western Blot, Rat)
• Sarah K Herring, Hee-Jung Moon, Punam Rawal, Anindit Chhibber, Liqin Zhao Brain clusterin protein isoforms and mitochondrial localization eLife 11/18/2019 [PMID: 31738162]
• Sarah K Herring, Hee-Jung Moon, Punam Rawal, Anindit Chhibber, Liqin Zhao Brain clusterin protein isoforms and mitochondrial localization eLife 2019-11-18 [PMID: 31738162] (Western Blot, Human)
• Kinchen J, Chen HH, Parikh K et al. Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease. Cell 9/25/2018 [PMID: 30270042]

Bioinformatics

• Gene Symbol: CLU
• Uniprot:
  • NP_001822()