Heat Shock Protein 70 (HSP70) ELISA Kit

Antigen: Heat Shock Protein 70 (HSP70)

Synonyms: 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

Reactivity: Rabbit

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN368548

Additional Information

Alternative name: Heat Shock Protein 70 (HSP-70)

Sample Type: Serum, Plasma

Specificity: This assay recognizes HSP-70. No significant cross-reactivity or interference was observed.

Sensitivity: The minimum detectable dose of HSP-70 is typically less than 0.156 ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest concentration that could be differentiated from zero. Detection Range: The minimum detectable dose of HSP-70 is typically less than 0.156 ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest concentration that could be differentiated from zero.

Synonyms: 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

Application Details

Principle: This assay employs the direct competitive inhibition enzyme immunoassay technique. An antibody specific for HSP-70 has been pre-coated onto a microplate. Add HSP-70 (Standards or samples) to the well, and then add Biotin-conjugated HSP-70. A competitive inhibition reaction is launched between HSP-70 (Standards or samples) and Biotin conjugated HSP-70 with the HSP-70 antibody. Then add the HRP-avidin to each well. The substrate solutions are added to the wells, respectively. And the color develops in opposite to the amount of HSP-70 bound in the initial step. The color development is stopped and the intensity of the color is measured.

Protocol: Reagent Preparation:
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 15 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 300 ml of Wash Buffer. Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.
Assay Procedure:
Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate. 1. Set a Blank well without any solutions. 2. Add 50 µ l of Standard or Sample to per well. 3. Add 50µl of Conjugate to each well (not to the Blank!). 4. Cover with the adhesive strip. Incubate for 60 minutes at 37° C. 5 5. Aspirate each well and wash, repeating the process for a total of three to five washes. Wash by filling each well with Wash Buffer (about 200µl) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. 6. Add 50µl of HRP-avidin to each well. Cover with the adhesive strip. Incubate for 30 minutes at 37°C. 7. Aspirate each well and wash as before. 8. Add 50µl of Substrate A and 50µl Substrate B to each well. Incubate for 10-30 minutes at 37°C. Keeping the plate away f rom drafts and other temperature fluctuations in the dark. 9. Add 50µl of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 10. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
Calculation of Results:
Average the duplicate readings for each standard, control, and sample and divide the average zero standard optical density. Create a standard curve by reducing the data using computer software. As an alternative, construct a standard curve by plotting the absorbance ratio for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve 6 through the points on the graph. The data may be linearized by plotting the log of the HSP-70 concentrations versus the ratio 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 diluent and repeat the assay. Any variation in 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.

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

Application Notes: Detection Wavelength: 450 nm

Components: Reagent (Quantity): Assay plate (96 tests) 1 Standard 5x1ml Conjugate (1x6ml), HRP-avidin (1x6ml), Wash Buffer (20×concentrate) (1 x15ml), Substrate A (1x6ml), Substrate B (1x6ml), Stop Solution (1x6ml)

Material not included: 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.

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.

Research Area: Metabolism, Heat Shock Proteins

Restrictions: For Research Use only