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Zebrafish HeatShock Protein 70 (HSP-70/HSPA9) ELISA Kit

SKU: orb1088148
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Description

The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to HeatShock Protein 70(HSP-70/HSPA9). Standards or samples are added to the appropriate microtiter plate wells then with a biotin-conjugated antibody specific to HeatShock Protein 70(HSP-70/HSPA9). Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain HeatShock Protein 70(HSP-70/HSPA9), biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of HeatShock Protein 70(HSP-70/HSPA9) in the samples is then determined by comparing the OD of the samples to the standard curve.

Research Area

Cancer Biology, Infectious Disease & Virology, Stem Cell & Developmental Biology

Images & Validation

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Application Notes
standard: 200 ng/mL. Test principle: The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Zebrafish HSP-70/HSPA9. Standards or samples are added to the appropriate microtiter plate wells then with a biotin-conjugated antibody specific to Zebrafish HSP-70/HSPA9. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Zebrafish HSP-70/HSPA9, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of Zebrafish HSP-70/HSPA9 in the samples is then determined by comparing the OD of the samples to the standard curve

Key Properties

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ReactivityZebrafish
Sample Typesserum, plasma, tissue homogenates, cell lysates, cell culture supernates and other biological fluids
Assay TypeSandwich
Assay Time3.5h
Range3.13-200 ng/mL
Sensitivity1.33 ng/mL
Concentration200 ng/mL

Procedure & Performance

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Assay Principle
The kit is based on a sandwich enzyme immunoassay principle. The microtiter plate is pre-coated with a capture antibody specific to the target analyte. Standards or samples are added to the wells, followed by a biotin-conjugated detection antibody specific for the analyte. Avidin conjugated to horseradish peroxidase (HRP) is then added and incubated. After addition of the TMB substrate, color develops only in wells containing the analyte bound to the detection antibody and HRP–avidin complex. The reaction is stopped with an acidic solution, and absorbance is measured at 450 nm ± 10 nm. The analyte concentration in the samples is determined by comparison with a standard curve.
Kit Components
1. ELISA Microplate
2. Standards
3. Detection Antibody
4. HRP-Streptavidin Conjugate
5. TMB Substrate
6. Dilution buffers
7. Stop Solution
8. Wash Buffer
9. Plate Sealers
10. Manual
Reagent Preparation
1. Wash Buffer: Prepare the 1X Wash Buffer using distilled water according to the manual.
2. Standard: Perform gradient dilution according to the instructions in the manual.
3. Other Concentrated Reagents: Dilute the concentrated reagents using the Dilution Buffers provided in the kit to 1 X working solutions as instructed in the manual. Always use a clean pipette tip for each different solution.
Assay Procedure
This procedure is for reference only.

1. After the kit equilibrates to room temperature, add standards or samples to each well and incubate.
2. Discard liquid, add wash buffer to each well, wash the plate three times, and blot dry on clean absorbent paper.
3. Add biotinylated antibody working solution to each well and incubate.
4. Discard liquid, add wash buffer to each well, wash the plate three times, and blot dry on clean absorbent paper.
5. Add streptavidin-HRP working solution to each well and incubate._x000b_6. Discard liquid, add wash buffer to each well, wash the plate five times, and blot dry on clean absorbent paper.
7. Add TMB substrate solution to each well and incubate in the dark.
8. Add stop solution to each well, mix thoroughly, and immediately read OD at 450 nm.
Materials Required
1. Microplate readers
2. Centrifuge
3. Incubator
4. Automated plate washer
5. Single-channel or multi-channel high-precision pipettes
6. Disposable pipette tips
7. Sterile tubes
8. Eppendorf tubes
9. Absorbent paper
10. Loading slots
Precision
Intra-assay Precision (Precision within an assay): CV% < 8%
Intra-assay precision was evaluated by testing multiple replicates of samples within the same plate.

Inter-assay Precision (precision between assays): CV% < 10%
Inter-assay precision was evaluated by testing samples across different plates.
Calculation of Results
1. Average the duplicate readings for each Standard, Control, and Sample, and subtract the mean optical density of the zero Standard.
2. Construct a standard curve by plotting the target concentration on the y-axis against absorbance on the x-axis and draw a curve through the data points.
3. Determine the sample concentration by substituting the OD450 value into the standard curve. For diluted samples, multiply the calculated value by the corresponding dilution factor.
Curve Fitting Softwares
1. Curve Expert
2. Thermo SkanIt RE
3. SciDAVis
4. LabPlot
5. ……

Storage & Handling

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StorageRefer to the Storage Guidelines in the Manual
Expiration DatePlease enquire.
DisclaimerFor research use only

Alternative Names

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DnaK, 70 Kilodalton Heat Shock Proteins, Heat shock 70 kDa protein 1, 2, heat shock 70kDa protein 1A, HSP70, HSP70 1, HSP70 1, HSP70 2, HSP70 1A, HSP70.1, HSP70.2, HSP70I, HSP72, HSPA1, HSPA1A, HSPA1B, HSP70, Heat Shock Protein 70
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Protocol Information

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48 T
$ 330.00
96 T
$ 470.00
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