Human Clusterin DuoSet ELISA

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Catalog # Availability Size / Price Qty
DY5874
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Human Clusterin ELISA Standard Curve
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Product Details
Procedure
Citations (6)
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Human Clusterin DuoSet ELISA Summary

Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Sample Volume Required
100 µL
Assay Range
62.5 - 4,000 pg/mL
Sufficient Materials
For fifteen 96-well plates*
Specificity
Please see the product datasheet

* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.

This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human Clusterin. The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet.

Product Features

  • Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
  • Development protocols are provided to guide further assay optimization
  • Assay can be customized to your specific needs
  • Economical alternative to complete kits

Kit Content

  • Capture Antibody
  • Detection Antibody
  • Recombinant Standard
  • Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)

Other Reagents Required

DuoSet Ancillary Reagent Kit 2 (5 plates): (Catalog # DY008) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 2.

The components listed above may be purchased separately:

PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered

Wash Buffer: (Catalog # WA126), or 0.05% Tween® 20 in PBS, pH 7.2-7.4

Reagent Diluent: (Catalog # DY995), or 1% BSA in PBS, pH 7.2-7.4, 0.2 µm filtered

Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)

Stop Solution: 2 N H2SO4 (Catalog # DY994)

Microplates: R&D Systems (Catalog # DY990)

Plate Sealers: ELISA Plate Sealers (Catalog # DY992)

Scientific Data

Human Clusterin ELISA Standard Curve

Product Datasheets

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Preparation and Storage

Shipping
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: Clusterin

Clusterin [also known as Apolipoprotein J, Sulfated Glycoprotein 2 (SGP-2), TRPM-2, and SP-40], is a secreted multifunctional 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, 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 approximately 80 kDa N-glycosylated and disulfide-linked heterodimer (7-9). Mature human Clusterin shares a 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 (10, 11). The ability of Clusterin to bind and neutralize non-oxidatively modified LDL reduces cytotoxicity in atherosclerotic plaques (12). The chaperone function of Clusterin helps to reduce the accumulation of beta -amyloid fibrils and damage due to amyloid plaques in Alzheimer's disease (13). An alternately spliced 50 kDa isoform of human Clusterin (nCLU) remains intracellular and is neither glycosylated nor cleaved into alpha and beta chains (5, 14). Cellular exposure to ionizing radiation promotes the translocation of nCLU to the nucleus where it interacts with Ku70 and promotes apoptosis (5, 14). This function contrasts with the cytoprotective effect of secreted Clusterin (15). During tumor progression, nCLU is down regulated while the secreted form is upregulated and may be aberrantly glycosylated (14, 16, 17). Increased circulating levels of Clusterin enhance tumor aggressiveness by inhibiting apoptosis and by promoting the epithelial to mesenchymal transition (18-20).

Entrez Gene IDs:
1191 (Human); 12759 (Mouse)
Alternate Names:
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

Assay Procedure

GENERAL ELISA PROTOCOL

Plate Preparation

  1. Dilute the Capture Antibody to the working concentration in PBS without carrier protein. Immediately coat a 96-well microplate with 100 μL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
  2. Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
  3. Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
  4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.

Assay Procedure

  1. Add 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
  2. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  3. Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
  4. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  5. Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  6. Repeat the aspiration/wash as in step 2.
  7. Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  8. Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  9. Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.

Citations for Human Clusterin DuoSet ELISA

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

6 Citations: Showing 1 - 6
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  1. Pre-analytical handling conditions and protein marker recovery from urine extracellular vesicles for bladder cancer diagnosis
    Authors: Lee, J;Kim, E;Park, J;Choi, S;Lee, MS;Park, J;
    PloS one
    Species: Human
    Sample Types: Extracellular Vesicles
  2. Expression and function of the luteinizing hormone choriogonadotropin receptor in human endometrial stromal cells
    Authors: ON Mann, CS Kong, ES Lucas, JJ Brosens, AC Hanyaloglu, PJ Brighton
    Scientific Reports, 2022-05-21;12(1):8624.
    Species: Human
    Sample Types: Cell Culture Supernates
  3. Serum and urinary biomarkers for early detection of acute kidney injury following Hypnale spp. envenoming
    Authors: ES Wijewickra, F Mohamed, IB Gawaramman, ZH Endre, NA Buckley, GK Isbister
    PloS Neglected Tropical Diseases, 2021-12-06;15(12):e0010011.
    Species: Human
    Sample Types: Urine
  4. Early identification of acute kidney injury in Russell's viper (Daboia russelii) envenoming using renal biomarkers
    Authors: I Ratnayake, F Mohamed, NA Buckley, IB Gawaramman, DM Dissanayak, U Chathurang, M Munasinghe, K Maduwage, S Jayamanne, ZH Endre, GK Isbister
    PLoS Negl Trop Dis, 2019-07-01;13(7):e0007486.
    Species: Human
    Sample Types: Urine
  5. Effects of freezer storage time on levels of complement biomarkers
    Authors: AR Morgan, C O'Hagan, S Touchard, S Lovestone, B Paul Morga
    BMC Res Notes, 2017-11-06;10(1):559.
    Species: Human
    Sample Types: Plasma
  6. Dexamethasone Modifies Cystatin C-Based Diagnosis of Acute Kidney Injury During Cisplatin-Based Chemotherapy
    Authors: TJ Pianta, JW Pickering, L Succar, M Chin, T Davidson, NA Buckley, F Mohamed, ZH Endre
    Kidney Blood Press. Res, 2017-03-17;42(1):62-75.
    Species: Human
    Sample Types: Urine

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