Heat Shock Protein 70 (HSP70) ELISA Kit

Antigen: Heat Shock Protein 70 (HSP70)

Synonyme: HSPA6, HSP70B', HSP70, heat shock protein 70, CHLOROPLAST HEAT SHOCK PROTEIN 70-2, CPHSC70-2EAT SHOCK PROTEIN 70-2, HEAT SHOCK PROTEIN 70, HEAT SHOCK PROTEIN 70-7, HSC70-7, K9P8.5, K9P8_5, F19K16.12, F19K16_12, LOC100305036, hsc70

Reaktivität: Huhn

Applikation: ELISA

Zertifikate: ISO 9001:2008

Produktnummer: ABIN364791

Produktbeschreibung

Produktmerkmale: This immunoassay kit allows for the in vitro quantitative determination of chicken HSP70 concentrations in cell culture supernates, serum, plasma and other biological fluids.

Weitere Bezeichnung: Heat Shock Protein 70 (Hsp-70)

Proben: Serum, Plasma

Beschreibung: The 70 kilodalton heat shock proteins (HSP70) are a family of ubiquitously expressed heat shock proteins. Proteins with similar structure exist in virtually all living organisms. The HSP70 are an important part of the cell's machinery for protein folding, and help to protect cells from stress. Members of the HSP70 family are strongly upregulated by heat stress and toxic chemicals, particularly heavy metals such as arsenic, cadmium, copper, mercury, etc. All of the HSP70 proteins have three major functional domains: an N-terminal ATPase domain, a substrate binding domain and a C-terminal domain rich in alpha helical structure. When an HSP70 protein is ATP bound, the lid is open and peptides bind and release relatively rapidly. When HSP70 proteins are ADP bound, the lid is closed, and peptides are tightly bound to the substrate binding domain. When not interacting with a substrate peptide, HSP70 is usually in an ATP bound state. HSP70 by itself is characterized by a very weak ATPase activity, such that spontaneous hydrolysis will not occur for many minutes. HSP70 also aids in transmembrane transport of proteins, by stabilizing them in a partially-folded state. HSP70 proteins can act to protect cells from thermal or oxidative stress. These stresses normally act to damage proteins, causing partial unfolding and possible aggregation. By temporarily binding to hydrophobic residues exposed by stress, HSP70 prevents these partially-denatured proteins from aggregating, and allows them to refold. Low ATP is characteristic of heat shock and sustained binding is seen as aggregation suppression, while recovery from heat shock involves substrate binding and nucleotide cycling. Finally, HSP70 seems to be able to participate in disposal of damaged or defective proteins. Interaction with CHIP (Carboxyl-terminus of HSP70 Interacting Protein)–an E3 ubiquitin ligase–allows HSP70 to pass proteins to the cell's ubiquitination and proteolysis pathways.

Spezifität: This assay recognizes recombinant and natural chicken HSP70. No significant cross-reactivity or interference was observed.

Sensitivität: The minimum detectable dose of chicken Hsp-70 is typically less than 0.12ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. Detection Range: The minimum detectable dose of chicken Hsp-70 is typically less than 0.12ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero.

Synonyme: HSPA6, HSP70B', HSP70, heat shock protein 70, CHLOROPLAST HEAT SHOCK PROTEIN 70-2, CPHSC70-2EAT SHOCK PROTEIN 70-2, HEAT SHOCK PROTEIN 70, HEAT SHOCK PROTEIN 70-7, HSC70-7, K9P8.5, K9P8_5, F19K16.12, F19K16_12, LOC100305036, hsc70

Anwendungen

Prinzip: The microtiter plate provided in this kit has been pre-coated with an antibody specific to HSP70. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for HSP70 and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3'5, 5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain HSP70, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of HSP70 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Protokoll: Preparation of Reagents: Bring all reagents to room temperature before use. 1. Wash Buffer If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 500 ml of Wash Buffer. 2. Standard Reconstitute the Standard with 1.0 ml of Sample Diluent. This reconstitution produces a stock solution of 5000 pg/ml. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (5000 pg/ml). The Sample Diluent serves as the zero standard (0 pg/ml). 3. Biotin-antibody Dilute to the working concentration specified on the vial label using Biotin-antibody Diluent(1:100), respectively. 4. HRP-avidin Dilute to the working concentration specified on the vial label using HRP-avidin Diluent(1:100), respectively. Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material. Sample Collection and Storage: Cell Culture Supernates Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles. Serum Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 g. Remove serum and assay immediately or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles. Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes of collection. Assay immediately or aliquot and store samples at -20°C. Avoid repeated freeze-thaw cycles. Assay Procedure: Calculation of Results: Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the HSP70 concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. Limitations of the Procedure: The kit should not be used beyond the expiration date on the kit label. Do not mix or substitute reagents with those from other lots or sources. It is important that the Calibrator Diluent selected for the standard curve be consistent with the samples being assayed. If samples generate values higher than the highest standard, dilute the samples with the appropriate Calibrator Diluent and repeat the assay. Any variation in Standard Diluent, operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding. This assay is designed to eliminate interference by soluble receptors, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.

Testdurchführung: Assay Time: 1-3h
Sample Volume: 50-100ul

Applikationshinweise: Detection Wavelength: 450 nm

Bestandteile: Reagent Quantity Assay plate 1 Standard 2 Sample Diluent 1 x 20 ml Biotin-antibody Diluent 1 x 10 ml HRP-avidin Diluent 1 x 10 ml Biotin-antibody 1 x 120µl HRP-avidin 1 x 120µl Wash Buffer 1 x 20 ml (25×concentrate) TMB Substrate 1 x 10 ml Stop Solution 1 x 10 ml STORAGE 1. Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag with desiccants to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date. 2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

Benötigtes Material: Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm. Pipettes and pipette tips. Deionized or distilled water. Squirt bottle, manifold dispenser, or automated microplate washer.

Lagerung: 1. Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag with desiccants to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date. 2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement. Expiration date: six months from the date of manufacture.

Forschungsgebiet: Metabolismus, Hitzeschockproteine

Beschränkungen: Nur für Forschungszwecke einsetzbar