Description
Human Clusterin (CLU) ELISA Kit
The Human Clusterin (CLU) ELISA Kit is a specialized assay designed for the precise measurement of clusterin levels in human biological samples including serum, plasma, and cell culture supernatants. This kit boasts exceptional sensitivity and specificity, ensuring dependable and consistent results for various research purposes.Clusterin, also known as apolipoprotein J, is a multifunctional protein involved in diverse biological processes such as cell aggregation, lipid transport, and apoptosis.
Its dysregulation has been implicated in various diseases including Alzheimer's disease, cancer, and kidney disease, underscoring its importance as a potential biomarker for disease diagnosis and therapeutic interventions.With the Human Clusterin ELISA Kit, researchers can accurately quantify clusterin levels and gain insights into its role in disease pathogenesis, paving the way for novel discoveries and advancements in biomedical research.
| Product Name: | Human Clusterin (CLU) ELISA Kit |
| SKU: | HUEB0252 |
| Size: | 96T |
| Target: | Human Clusterin (CLU) |
| Synonyms: | Aging-associated gene 4 protein, Apolipoprotein J, Complement cytolysis inhibitor, Complement-associated protein SP-40, 40, Ku70-binding protein 1, NA1/NA2, Sulfated glycoprotein 2, Testosterone-repressed prostate message 2, Apo-J, CLI, SGP-2, TRPM-2, AAG4, APOJ, CLI, KUB1 |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 0.78-50ng/mL |
| Sensitivity: | 0.38ng/mL |
| Intra CV: | 3.7% | ||||||||||||||||||||
| Inter CV: | 7.3% | ||||||||||||||||||||
| Linearity: |
| ||||||||||||||||||||
| Recovery: |
| ||||||||||||||||||||
| Function: | Isoform 4: Does not affect caspase or BAX-mediated intrinsic apoptosis and TNF-induced NF-kappa-B-activity (PubMed:24073260). Promotes cell death through interaction with BCL2L1 that releases and activates BAX (PubMed:21567405). |
| Uniprot: | P10909 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human Clusterin |
| Sub Unit: | Antiparallel disulfide-linked heterodimer of an alpha chain and a beta chain (PubMed:12047389, PubMed:1491011, PubMed:1551440, PubMed:2780565, PubMed:2387851, PubMed:8328966, PubMed:1974459). Self-associates and forms higher oligomers (PubMed:1903064). Interacts with a broad range of misfolded proteins, including APP, APOC2 and LYZ (PubMed:17407782, PubMed:8328966, PubMed:17412999). Slightly acidic pH promotes interaction with misfolded proteins (PubMed:12176985). Forms high-molecular weight oligomers upon interaction with misfolded proteins (PubMed:19535339). Interacts with APOA1, LRP2, CLUAP1 and PON1 (PubMed:15480429, PubMed:17260971, PubMed:1742316, PubMed:8292612, PubMed:1903064). Interacts with the complement complex (PubMed:2601725). Interacts (via alpha chain) with XRCC6 (By similarity). Interacts with SYVN1, COMMD1, BTRC, CUL1 and with ubiquitin and SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complexes (PubMed:17451556, PubMed:20068069). Interacts (via alpha chain) with BAX in stressed cells, where BAX undergoes a conformation change leading to association with the mitochondrial membrane (PubMed:16113678). Does not interact with BAX in unstressed cells (PubMed:16113678). Found in a complex with LTF, CLU, EPPIN and SEMG1 (PubMed:17567961). Interacts (immaturely glycosylated pre-secreted form) with HSPA5; this interaction promotes CLU stability and facilitates stress-induced CLU retrotranslocation from the secretory pathway to the mitochondria, thereby reducing stress-induced apoptosis by stabilizing mitochondrial membrane integrity (PubMed:22689054). Interacts (isoform 4) with BCL2L1; this interaction releases and activates BAX and promotes cell death (PubMed:21567405). Interacts with TGFBR2 and ACVR1 (PubMed:8555189). Interacts (secreted form) with STMN3; this interaction may act as an important modulator during neuronal differentiation (By similarity). |
| Research Area: | Cardiovascular |
| Subcellular Location: | Nucleus Cytoplasm Mitochondrion membrane Peripheral membrane protein Cytoplasmic side Cytoplasm Cytosol Microsome Endoplasmic reticulum Mitochondrion Mitochondrion membrane Cytoplasm Perinuclear region Cytoplasmic vesicle Secretory vesicle Chromaffin granule Secreted isoforms can retrotranslocate from the secretory compartments to the cytosol upon cellular stress (PubMed:17451556). Detected in perinuclear foci that may be aggresomes containing misfolded, ubiquitinated proteins (PubMed:20068069). Detected at the mitochondrion membrane upon induction of apoptosis (PubMed:17689225). Under ER stress, a immaturely glycosylated pre-secreted form retrotranslocates from the endoplasmic reticulum (ER)-Golgi network to the cytoplasm to localize in the mitochondria through HSPA5 interaction (PubMed:22689054). ER stress reduces secretion (PubMed:22689054). Under the stress, minor amounts of non-secreted forms accumulate in cytoplasm (PubMed:24073260, PubMed:22689054, PubMed:17451556). Non-secreted forms emerge mainly from failed translocation, alternative splicing or non-canonical initiation start codon (PubMed:24073260, PubMed:12551933). |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | CLU: Isoform 1 functions as extracellular chaperone that prevents aggregation of nonnative proteins. Prevents stress- induced aggregation of blood plasma proteins. Inhibits formation of amyloid fibrils by APP, APOC2, B2M, CALCA, CSN3, SNCA and aggregation-prone LYZ variants (in vitro). Does not require ATP. Maintains partially unfolded proteins in a state appropriate for subsequent refolding by other chaperones, such as HSPA8/HSC70. Does not refold proteins by itself. Binding to cell surface receptors triggers internalization of the chaperone-client complex and subsequent lysosomal or proteasomal degradation. Secreted isoform 1 protects 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. Promotes proteasomal degradation of COMMD1 and IKBKB. Modulates NF-kappa-B transcriptional activity. Nuclear isoforms promote apoptosis. Mitochondrial isoforms suppress BAX-dependent release of cytochrome c into the cytoplasm and inhibit apoptosis. Plays a role in the regulation of cell proliferation. Belongs to the clusterin family. 5 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Secreted; Apoptosis; Mitochondrial; Secreted, signal peptide Chromosomal Location of Human Ortholog: 8p21-p12 Cellular Component: extracellular matrix; Golgi apparatus; extracellular space; mitochondrion; endoplasmic reticulum; perinuclear region of cytoplasm; mitochondrial membrane; cytoplasm; extracellular region; nucleus; cytosol Molecular Function:protein binding; chaperone binding; ubiquitin protein ligase binding; ATPase activity; misfolded protein binding Biological Process: platelet activation; release of cytochrome c from mitochondria; response to misfolded protein; positive regulation of nitric oxide biosynthetic process; protein stabilization; positive regulation of apoptosis; response to virus; cell morphogenesis; microglial cell activation; positive regulation of tumor necrosis factor production; negative regulation of protein homooligomerization; activation of NF-kappaB transcription factor; complement activation; reverse cholesterol transport; platelet degranulation; myelin maintenance in the central nervous system; positive regulation of proteasomal ubiquitin-dependent protein catabolic process; protein import; innate immune response; lipid metabolic process; blood coagulation; chaperone-mediated protein complex assembly; complement activation, classical pathway |
| NCBI Summary: | The protein encoded by this gene is a secreted chaperone that can under some stress conditions also be found in the cell cytosol. It has been suggested to be involved in several basic biological events such as cell death, tumor progression, and neurodegenerative disorders. Alternate splicing results in both coding and non-coding variants.[provided by RefSeq, May 2011] |
| UniProt Code: | P10909 |
| NCBI GenInfo Identifier: | 116533 |
| NCBI Gene ID: | 1191 |
| NCBI Accession: | P10909.1 |
| UniProt Secondary Accession: | P10909,P11380, P11381, Q2TU75, Q5HYC1, Q7Z5B9, B2R9Q1 B3KSE6, |
| UniProt Related Accession: | P10909,AAB25217 |
| Molecular Weight: | Calculated: 32kDa/48kDa/52kDa/53kDa/57kDaObserved: 40kDa |
| NCBI Full Name: | Clusterin |
| NCBI Synonym Full Names: | clusterin |
| NCBI Official Symbol: | CLU |
| NCBI Official Synonym Symbols: | CLI; AAG4; APOJ; CLU1; CLU2; KUB1; SGP2; APO-J; SGP-2; SP-40; TRPM2; TRPM-2; NA1/NA2 |
| NCBI Protein Information: | clusterin; apolipoprotein J; ku70-binding protein 1; sulfated glycoprotein 2; aging-associated protein 4; complement lysis inhibitor; complement cytolysis inhibitor; complement-associated protein SP-40,40; testosterone-repressed prostate message 2 |
| UniProt Protein Name: | Clusterin |
| UniProt Synonym Protein Names: | Aging-associated gene 4 protein; Apolipoprotein J; Apo-J; Complement cytolysis inhibitor; CLI; Complement-associated protein SP-40,40; Ku70-binding protein 1; NA1/NA2; Testosterone-repressed prostate message 2; TRPM-2 |
| Protein Family: | Clusterin |
| UniProt Gene Name: | CLU |
| UniProt Entry Name: | CLUS_HUMAN |
| Component | Quantity (96 Assays) | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | -20°C |
| Lyophilized Standard | 2 | -20°C |
| Sample Diluent | 20ml | -20°C |
| Assay Diluent A | 10mL | -20°C |
| Assay Diluent B | 10mL | -20°C |
| Detection Reagent A | 120µL | -20°C |
| Detection Reagent B | 120µL | -20°C |
| Wash Buffer | 30mL | 4°C |
| Substrate | 10mL | 4°C |
| Stop Solution | 10mL | 4°C |
| Plate Sealer | 5 | - |
Other materials and equipment required:
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Incubator
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
| Step | |
| 1. | Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C. |
| 2. | Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform. |
| 3. | Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper. |
| 4. | Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C. |
| 5. | Repeat the wash process for five times as conducted in step 3. |
| 6. | Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction. |
| 7. | Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. |
| 8. | Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters. |
| 9. | After experiment, store all reagents according to the specified storage temperature respectively until their expiry. |
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
| Sample Type | Protocol |
| Serum | If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. |
| Plasma | Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit. |
| Urine & Cerebrospinal Fluid | Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid. |
| Cell culture supernatant | Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately. |
| Cell lysates | Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Tissue homogenates | The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C. |
| Tissue lysates | Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Breast Milk | Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles. |
