Human HSP60 ELISA Kit
- SKU:
- HUFI00811
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P10809
- Sensitivity:
- 0.938ng/ml
- Range:
- 1.563-100ng/ml
- ELISA Type:
- Sandwich
- Synonyms:
- HSP-60, HSPD1, Heat Shock Protein 60, cpn60, GROEL, SPG13, 60 kDa chaperonin, Chaperonin 60, heat shock 60kD protein 1, chaperonin, Heat shock protein 60, heat shock protein 65, HLD4, Hsp60, HSP65, HuCHA60, P60 lymphocyte protein, short heat shock pr
- Reactivity:
- Human
- Research Area:
- Immunology
Description
Human HSP60 ELISA Kit
Heat shock protein 60 (HSP60) is a member of the heat shock protein family. It is highly expressed in the brain, heart, and skeletal muscle. It is also found in other organs such as the kidney and liver. Heat shock protein 60 (HSP60) is a molecular chaperone that plays a key role in the prevention and repair of protein damage. It functions in the endoplasmic reticulum, where it is used as a marker for the presence of unfolded proteins. HSP60 binds to damaged proteins, preventing them from aggregating into amyloid plaques and spreading to other cells.
| Product Name: | Human HSP60 ELISA Kit |
| Product Code: | HUFI00811 |
| Size: | 96 Assays |
| Alias: | HSP-60, HSPD1, Heat Shock Protein 60, cpn60, GROEL, SPG13, 60 kDa chaperonin, Chaperonin 60, heat shock 60kD protein 1, chaperonin, Heat shock protein 60, heat shock protein 65, HLD4, Hsp60, HSP65, HuCHA60, P60 lymphocyte protein, short heat shock protein 60 Hsp60s1, spastic paraplegia 13, autosomal dominant |
| Detection method: | Sandwich ELISA, Double Antibody |
| Application: | This immunoassay kit allows for the in vitro quantitative determination of Human HSPD1/HSP-60 concentrations in serum plasma and other biological fluids. |
| Sensitivity: | 0.938ng/ml |
| Range: | 1.563-100ng/ml |
| Storage: | 4°C for 6 months |
| Note: | For Research Use Only |
| Recovery: | Matrices listed below were spiked with certain level of Human HSPD1/HSP-60 and the recovery rates were calculated by comparing the measured value to the expected amount of Human HSPD1/HSP-60 in samples. | ||||||||||||||||
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| Linearity: | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human HSPD1/HSP-60 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. | ||||||||||||||||
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| CV(%): | Intra-Assay: CV<8% Inter-Assay: CV<10% |
| Component | Quantity | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | 4°C for 6 months |
| Lyophilized Standard | 2 | 4°C/-20°C |
| Sample/Standard Dilution Buffer | 20ml | 4°C |
| Biotin-labeled Antibody(Concentrated) | 120ul | 4°C (Protect from light) |
| Antibody Dilution Buffer | 10ml | 4°C |
| HRP-Streptavidin Conjugate(SABC) | 120ul | 4°C (Protect from light) |
| SABC Dilution Buffer | 10ml | 4°C |
| TMB Substrate | 10ml | 4°C (Protect from light) |
| Stop Solution | 10ml | 4°C |
| Wash Buffer(25X) | 30ml | 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
| Uniprot | P10809 |
| UniProt Protein Function: | HSP60: Implicated in mitochondrial protein import and macromolecular assembly. May facilitate the correct folding of imported proteins. May also prevent misfolding and promote the refolding and proper assembly of unfolded polypeptides generated under stress conditions in the mitochondrial matrix. Interacts with HRAS. Interacts with HBV protein X and HTLV-1 protein p40tax. Belongs to the chaperonin (HSP60) family. |
| UniProt Protein Details: | Protein type:Chaperone; Mitochondrial Chromosomal Location of Human Ortholog: 2q33.1 Cellular Component: extracellular space; cell surface; protein complex; mitochondrion; early endosome; coated pit; cytosol; secretory granule; membrane; mitochondrial matrix; cytoplasm; mitochondrial inner membrane; plasma membrane; lipopolysaccharide receptor complex; cyclin-dependent protein kinase activating kinase holoenzyme complex Molecular Function:protein binding; p53 binding; lipopolysaccharide binding; ubiquitin protein ligase binding; chaperone binding; ATPase activity; double-stranded RNA binding; unfolded protein binding; DNA replication origin binding; single-stranded DNA binding; ATP binding Biological Process: B cell proliferation; caspase activation; B cell activation; T cell activation; protein stabilization; viral reproduction; positive regulation of apoptosis; positive regulation of interleukin-12 production; protein maturation; isotype switching to IgG isotypes; positive regulation of interleukin-6 production; B cell cytokine production; positive regulation of interleukin-10 production; response to unfolded protein; MyD88-dependent toll-like receptor signaling pathway; positive regulation of interferon-gamma production; 'de novo' protein folding; protein refolding; positive regulation of T cell activation; positive regulation of T cell mediated immune response to tumor cell; chaperone-mediated protein complex assembly; positive regulation of interferon-alpha production; negative regulation of apoptosis; positive regulation of macrophage activation Disease: Spastic Paraplegia 13, Autosomal Dominant; Leukodystrophy, Hypomyelinating, 4 |
| NCBI Summary: | This gene encodes a member of the chaperonin family. The encoded mitochondrial protein may function as a signaling molecule in the innate immune system. This protein is essential for the folding and assembly of newly imported proteins in the mitochondria. This gene is adjacent to a related family member and the region between the 2 genes functions as a bidirectional promoter. Several pseudogenes have been associated with this gene. Two transcript variants encoding the same protein have been identified for this gene. Mutations associated with this gene cause autosomal recessive spastic paraplegia 13. [provided by RefSeq, Jun 2010] |
| UniProt Code: | P10809 |
| NCBI GenInfo Identifier: | 129379 |
| NCBI Gene ID: | 3329 |
| NCBI Accession: | P10809.2 |
| UniProt Secondary Accession: | P10809,Q38L19, Q9UCR6, B2R5M6, B7Z712, |
| UniProt Related Accession: | P10809 |
| Molecular Weight: | 17,100 Da |
| NCBI Full Name: | 60 kDa heat shock protein, mitochondrial |
| NCBI Synonym Full Names: | heat shock 60kDa protein 1 (chaperonin) |
| NCBI Official Symbol: | HSPD1 |
| NCBI Official Synonym Symbols: | HLD4; CPN60; GROEL; HSP60; HSP65; SPG13; HSP-60; HuCHA60 |
| NCBI Protein Information: | 60 kDa heat shock protein, mitochondrial; chaperonin 60; 60 kDa chaperonin; heat shock protein 65; P60 lymphocyte protein; mitochondrial matrix protein P1; short heat shock protein 60 Hsp60s1 |
| UniProt Protein Name: | 60 kDa heat shock protein, mitochondrial |
| UniProt Synonym Protein Names: | 60 kDa chaperonin; Chaperonin 60; CPN60; Heat shock protein 60; HSP-60; Hsp60; HuCHA60; Mitochondrial matrix protein P1; P60 lymphocyte protein |
| Protein Family: | 60 kDa heat shock protein |
| UniProt Gene Name: | HSPD1 |
| UniProt Entry Name: | CH60_HUMAN |
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.
| Step | Protocol |
| 1. | Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells! |
| 2. | Aliquot 0.1ml standard solutions into the standard wells. |
| 3. | Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well. |
| 4. | Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells. |
| 5. | Seal the plate with a cover and incubate at 37 °C for 90 min. |
| 6. | Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2. |
| 7. | Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall. |
| 8. | Seal the plate with a cover and incubate at 37°C for 60 min. |
| 9. | Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash. |
| 10. | Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min. |
| 11. | Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min. |
| 12. | Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color. |
| 13. | Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately. |
| 14. | Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution. |
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. |
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