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This kit can only be used for scientific research, not for medical diagnosis
Rat (Rat) Heart-type Fatty Acid Binding Protein (h-FABP) ELISA Test Kit Instructions
Detection principle
The kit uses a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). Forward
First coat the heart-shaped fatty acid binding protein (h-FABP) antibody in the coated microwells, add
Incorporate specimens, standards, HRP-labeled detection antibodies, incubate and wash thoroughly. Bottom
TMB develops color, which is converted into blue under the catalysis of peroxidase, and in the role of acid
It turns into the final yellow. The shade of color and the heart-shaped fatty acid binding protein in the sample
(h-FABP) was positively correlated. Measure the absorbance (OD) at 450nm with a microplate reader
Value), calculate the sample concentration.
Sample collection, processing and storage methods
1. Serum: use a test tube free of pyrogens and endotoxins, avoid any cells during operation
After stimulating and collecting blood, centrifuge at 3000 rpm for 10 minutes to quickly and carefully serum and red blood cells
Separate.
2. Plasma: EDTA, citrate or heparin anticoagulation. Take the supernatant by centrifugation at 3000 rpm for 30 minutes.
3. Cell supernatant: centrifuge at 3000 rpm for 10 minutes to remove particles and polymers.
4. Tissue homogenate: Add tissue to the right amount of saline and mash. Centrifuge at 3000 rpm for 10 minutes
Take the supernatant.
5. Preservation: If the samples are not tested in time after collection, please aliquot according to the dosage and freeze in
-20 ℃, avoid repeated freezing and thawing, thaw at room temperature and ensure that the sample is thawed evenly and fully.
Bring your own items
1. Microplate reader (450nm)
2. High-precision sampler and tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL
3. 37 ℃ thermostat
Operation notes
1. Store the kit at 2-8 ° C and equilibrate at room temperature for 20 minutes before use. Removed from the refrigerator
The concentrated washing liquid will have crystals, which is normal. Heating the water bath will completely dissolve the crystals
Use it later. 2. The slats not used in the experiment should be immediately returned to the ziplock bag, sealed (dry at low temperature) and stored.
3. S0 standard with a concentration of 0 can be regarded as a negative control or blank; follow the instructions
The sample has been diluted 5 times during the book operation, and the final result is multiplied by 5 to obtain the actual sample concentration.
4. Perform the incubation operation strictly in accordance with the time, amount of liquid and sequence indicated in the manual.
5. Shake all liquid components thoroughly before use.
Kit composition
Name 96-well configuration 48-well configuration remarks
Microwell microplate 12 well × 8 strips 12 well × 4 strips None
Standard product 0.3mL * 6 tube 0.3mL * 6 tube
Sample diluent 6mL 3mL None
Detection antibody-HRP 10mL 5mL None
20 × Washing buffer 25mL 15mL Dilute according to the instructions
Substrate A 6mL 3mL None
Substrate B 6mL 3mL None
Stop solution 6mL 3mL None
Sealing film 2 sheets 2 sheets without
Instructions 1 copy 1 copy
1 ziplock bag 1 no
Note: The concentration of standard products (S0-S5) is: 0, 75, 150, 300, 600, 1200 pg / mL
Reagent preparation
Dilution of 20 × washing buffer: 1:20 dilution of distilled water, ie 1 part of 20 × washing buffer
Add 19 parts of distilled water.
Washing method
1. Manually wash the plate: throw away all the liquid in the hole, fill each hole with washing liquid, let it rest for 1min
Dip the liquid in the hole on the absorbent paper and wash the plate 5 times.
2. Automatic plate washing machine: Inject 350μL of washing solution into each well, soak for 1min, wash the plate 5 times.
Steps
1. Remove the required slats from the aluminum foil bag after 20 minutes of room temperature equilibration, and the remaining slats shall be self-sealed
The bag is sealed and put back at 4 ° C.
2. Set up standard wells and sample wells, add 50μL of standard products of different concentrations to the standard wells;
3. First add 10μL of the sample to be tested to the sample well, then add 40μL of the sample diluent; the blank well does not
plus.
4. In addition to blank wells, add horseradish peroxidase to each of the standard wells and sample wells
(HRP) 100 μL of detection antibody, seal the reaction well with a plate sealer, and 37 ° C water bath
Incubate in a pot or incubator for 60 minutes.
5. Discard the liquid, pat dry on absorbent paper, fill each well with washing liquid, let stand for 1min, shake off the washing liquid, pat dry on absorbent paper, and repeat washing the plate 5 times (you can also wash the plate with a washing machine).
6. Add 50 μL of substrate A and B to each well, and incubate at 37 ° C in the dark for 15 minutes.
7. Add 50μL of stop solution to each well, and measure the wells at 450nm wavelength within 15min
OD value.
Result judgment
Draw the standard curve: In the Excel worksheet, take the concentration of the standard as the abscissa, corresponding
The OD value is used as the ordinate, and the linear regression curve of the standard product is drawn, and various samples are calculated according to the curve equation
This concentration value.
Kit performance
1. Accuracy: the correlation coefficient R between the linear regression of the standard product and the expected concentration is greater than or equal to
0.9900.
2. Sensitivity: The minimum detection concentration is less than 10 pg / mL.
3. Specificity: Does not cross-react with other soluble structural analogs.
4. Repeatability: The coefficients of variation within and between panels are less than 15%.
5. Storage: 2-8 ℃, protected from light and moisture.
6. Validity: 6 months
Disclaimer
1. The kit is for research use only and should not be used in clinical or human experiments, otherwise
The experimenter shall bear all the consequences, and our company shall not be responsible.
2. Strictly follow the instructions, the experimenter violates the instructions, and the consequences will be determined by the experimenter
bear.
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