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|Sample type||Average %||Range|
|Cell culture supernatant||108||104% - 113%|
|Serum||95||91% - 99%|
|Heparin Plasma||85||83% - 87%|
|EDTA Plasma||92||86% - 96%|
Clusterin in vitro SimpleStep ELISA® (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of Clusterin protein in human plasma, serum and cell culture supernatants.
The SimpleStep ELISA® employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.
Clusterin (Apolipoprotein J) is composed of an antiparallel disulfide-linked heterodimer of an alpha chain and a beta chain that self-associate and can form higher oligomers. It is ubiquitously expressed in various tissues. Clusterin functions as an extracellular chaperone that prevents aggregation of nonnative proteins. It does not require ATP and does not refold proteins by itself. Clusterin does so by maintaining partially unfolded proteins in a state appropriate for subsequent refolding by other chaperones, such as HSPA8/HSC70. Clusterin prevents stress-induced aggregation of blood plasma proteins by inhibiting the formation of amyloid fibrils through APP, APOC2, B2M, CALCA, CSN3, SNCA and aggregation-prone LYZ variants (in vitro). It can bind to cell surface receptors and trigger internalization of the chaperone-client complex and subsequent lysosomal or proteasomal degradation.
Secreted isoforms protect cells against apoptosis and against cytolysis by complement. Intracellular isoforms interact with ubiquitin and SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complexes and promote the ubiquitination and subsequent proteasomal degradation of target proteins. Clusterin can retrotranslocate from the secretory compartments to the cytosol upon induction of stress. Nuclear isoforms promote apoptosis. Mitochondrial isoforms suppress BAX-dependent release of cytochrome c into the cytoplasm and inhibit apoptosis.
|Components||1 x 96 tests|
|10X Human Clusterin Capture Antibody||2 x 300µl|
|10X Human Clusterin Detector Antibody||2 x 300µl|
|10X Wash Buffer PT (ab206977)||1 x 20ml|
|Antibody Diluent CPI - HAMA Blocker (ab193969)||1 x 6ml|
|Human Clusterin Lyophilized Purified Protein||2 vials|
|Plate Seals||1 unit|
|Sample Diluent NS (ab193972)||1 x 50ml|
|SimpleStep Pre-Coated 96-Well Microplate (ab206978)||1 unit|
|Stop Solution||1 x 12ml|
|TMB Substrate||1 x 12ml|
Our Abpromise guarantee covers the use of ab174447 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Sandwich ELISA||Use at an assay dependent concentration.|
Background-subtracted data values (mean +/- SD) are graphed.
Mean human Clusterin is 63 µg/mL and values fall within expected normal reference ranges.
The concentrations of Clusterin were measured in duplicates, interpolated from the Clusterin standard curves and corrected for sample dilution. Undiluted samples are as follows: serum 0.01%, plasma (EDTA) 0.01%, plasma (heparin) 0.005%. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean Clusterin concentration was determined to be 106 ug/mL in serum, 118 ug/mL in plasma (EDTA), and 200 ug/mL in plasma (heparin)
HeLa cells (1x106 cells/mL) were cultured in RPMI media supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin sulfate. Conditioned media was harvested after 48 hours, aliquoted and assayed for endogenous Clusterin levels. Background subtracted data from triplicate measurements are plotted. Mean calculated value for endogenous Clusterin was 231 ng/mL.
ab174447 has not yet been referenced specifically in any publications.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"