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Mouse Glucose Kinase (GCK) ELISA Kit Analysis Instructions

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本试剂盒用于体外定量检测血清、血浆、组织、细胞上清及相关液体样本中 小鼠葡萄糖激酶 GCK )的含量。 l This kit is used for the quantitative determination of mouse glucokinase ( GCK ) in serum, plasma, tissue, cell supernatant and related liquid samples in vitro .
有效期:6个月 l Validity: 6 months
保存条件: 2 -8℃ lStorage conditions: 2 -8 ℃
本试剂盒仅供体外研究使用,不用于临床诊断 l This kit is for in vitro research use only, not for clinical diagnosis
 
Experimental principle
The kit uses a double antibody one-step sandwich method for enzyme-linked immunosorbent assay (ELISA). To the coated microwells pre-coated with mouse glucokinase (GCK) capture antibody, add the sample, standard, and HRP-labeled detection antibody in order, incubate and wash thoroughly. The color is developed with the substrate TMB, and TMB is converted to blue by peroxidase catalysis, and is converted to the final yellow by the action of acid. The shade of color is positively correlated with the mouse glucokinase (GCK) in the sample. Measure the absorbance (OD value) at 450nm with a microplate reader to calculate the sample concentration.
 
Sample processing and requirements
:全血标本请于室温放置2小时或4℃过夜后于1000g离心20分钟,取上清即可检测,或将标本放于-20℃或-80℃保存,但应避免反复冻融。 1. Serum : Whole blood samples should be left at room temperature for 2 hours or 4 C overnight. Centrifuge at 1000g for 20 minutes, and then take the supernatant to test. Or store the samples at -20 C or -80 C, but avoid repeated freezing. melt.
:可用EDTA或肝素作为抗凝剂,标本采集后30分钟内于2 - 8C 1000g离心20分钟,或将标本放于-20℃或-80℃保存,但应避免反复冻融。 2. Plasma : EDTA or heparin can be used as an anticoagulant. Centrifuge at 1000 g at 2-8 C for 20 minutes within 30 minutes after collection, or store the sample at -20 C or -80 C, but avoid repeated freeze-thaw cycles. .
:用预冷的PBS (0.01M, pH=7.4)冲洗组织,去除残留血液(匀浆中裂解的红细胞会影响测量结果),称重后将组织剪碎。 3. Tissue homogenate : Rinse the tissue with pre-chilled PBS (0.01M, pH = 7.4) to remove residual blood (the lysed red blood cells in the homogenate will affect the measurement result), and cut the tissue after weighing. The cut tissue and the corresponding volume of PBS (generally based on a weight to volume ratio of 1: 9, such as 1g of tissue sample corresponds to 9mL of PBS, the specific volume can be adjusted according to the needs of the experiment, and make a record. It is recommended to add in PBS Protease inhibitors) were added to a glass homogenizer and ground thoroughly on ice. To further lyse the tissue cells, the homogenate can be sonicated or repeatedly freeze-thaw. Finally, the homogenate was centrifuged at 5000 g for 5-10 minutes, and the supernatant was taken for detection.
:1000g离心20分钟,取上清即可检测,或将标本放于-20℃或-80℃保存,但应避免反复冻融。 4. Cell culture supernatant or other biological specimens : Centrifuge at 1000g for 20 minutes, and take the supernatant for detection, or store the specimens at -20 C or -80 C, but avoid repeated freeze-thaw cycles.
Note: The hemolysis of the specimen will affect the final test results, so this test should not be performed on hemolyzed specimens.

Specimen processing
1. The company is only responsible for the kit itself, not for sample consumption caused by using the kit. Users are requested to fully consider the possible use of the sample before use, and reserve sufficient samples.
2. The content of the specimen should be predicted before the experiment. If the concentration of the specimen is too high, the specimen should be diluted so that the diluted specimen conforms to the detection range of the kit and then multiplied by the corresponding dilution factor during calculation. Specimens were diluted with 0.01mol / L PBS (PH = 7.0-7.2).
3. If the tested samples are not included in the samples listed in the instruction manual, it is recommended to conduct pre-experiments to verify their effectiveness, and pay attention to retaining the samples.
4. Tissue homogenates or cell extracts prepared using chemical lysates may cause deviations in ELISA results due to the introduction of certain chemicals.
5. If the sample is a cell culture supernatant, it may not be detected because there are many interference factors in this type of sample, such as: cell status, cell number, and sampling time.
6. Certain natural or recombinant proteins, including prokaryotic and eukaryotic recombinant proteins, may not be detected because they do not match the detection and capture antibodies used in this product.
7. It is recommended to use fresh samples. Excessive storage time may result in protein degradation or denaturation, which may lead to deviations in experimental results.
 
Required and not supplied reagents and equipment
Microplate reader (450nm)
2. High-precision sampler and tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL
3. 37 ℃ constant temperature box
4. Distilled or deionized water
 
Kit composition
name
96-well configuration
48-hole configuration
Remark
Microtiter plate
8 holes 12
8 holes 6
no
Standard
0.3mL * 6 tube
0.3mL * 6 tube
no
Sample diluent
6mL
3mL
no
Detection antibody-HRP
10mL
5mL
no
20 washing buffer
25mL
15mL
Dilute according to instructions
Substrate A
6mL
3mL
no
Substrate B
6mL
3mL
no
Stop solution
6mL
3mL
no
Sealing film
2 pieces
2 pieces
no
Manual
1 serving
1 serving
no
Ziplock bag
1
1
no
Remarks :
1. The standard concentration is: 80, 40, 20, 10, 5, 0 U / L
2. After a large number of normal specimens are tested, the normal concentration values of the specimens are within the detection range provided by the kit, and 50 μL samples can be directly loaded during the experiment. When some sample values exceed the maximum standard concentration, the sample dilution solution can be used to properly dilute the sample before the experiment.
 
Precautions
1. Incubate strictly according to the specified time and temperature to ensure accurate results. All reagents must reach room temperature 20-25 C before use. Keep reagents refrigerated immediately after use.
2. Incorrect plate washing can lead to inaccurate results. Be sure to suck up as much liquid as possible before adding the substrate. Do not allow microwells to dry out during incubation.
3. Eliminate the residual liquid and fingerprints on the bottom of the board, otherwise it will affect the OD value.
4. The substrate solution should be colorless or very light. The substrate solution that has turned blue cannot be used.
5. Avoid cross-contamination of reagents and specimens to avoid false results.
6. Avoid direct light exposure during storage and incubation.
7. After equilibrating to room temperature, open the sealed bag to prevent water droplets from condensing on the cold slat.
8. No reaction reagent should come into contact with bleaching solvents or strong gases emitted by bleaching solvents. Any bleaching ingredient will destroy the biological activity of the reagents in the kit.
9. Do not use expired products.
如果可能传播疾病,所有的样品都应管理好,按照规定的程序处理样品和检测装置10. If the disease is likely to be transmitted, all samples should be managed, and the samples and testing equipment should be handled in accordance with the prescribed procedures .
 
Reagent preparation
Remove the kit from the refrigerated environment and allow it to equilibrate at room temperature before use.
20 washing buffer dilution: Distilled water is diluted 1:20, ie 1 part of 20 washing buffer plus 19 parts of distilled water.
 
Steps
1. Take out the required slat from the aluminum foil bag after equilibrating at room temperature for 20 minutes, and seal the remaining slab with ziplock bag and return it to 4 C.
2. Set up standard wells and sample wells, and add 50 μL of standards at different concentrations to the standard wells;
3. Add 50 μL of the sample to be tested into the sample well; blank wells are not added.
4. In addition to blank wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each well of the standard and sample wells. Seal the reaction wells with a sealing plate and incubate in a 37 C water bath or incubator. 60min.
5. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350 μL), let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat washing the plate 5 times (also can be washed with a washing machine board).
6. Add 50 μL each of substrates A and B to each well, and incubate at 37 C in the dark for 15 minutes.
7. Add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 minutes.
 
Calculation of experimental results
Take the measured OD value of the standard product as the abscissa, and the concentration value of the standard product as the ordinate, draw a standard curve on the coordinate paper or use related software, and obtain a linear regression equation. Substitute the OD value of the sample into the equation to calculate the sample concentration.
 
Kit performance
1. Detection range: 2.5 U / L-80 U / L.
2. Sensitivity: The minimum detection concentration is less than 0.1 U / L.
3. Specificity: Does not cross-react with other soluble structural analogs.
4. Repeatability: the coefficient of variation within the plate is less than 10%, and the coefficient of variation between the plates is less than 15%.
 
Description
1. Due to the current conditions and scientific and technological level, it is not yet possible to conduct a comprehensive identification and analysis of all raw materials provided by all suppliers, this product may have certain quality and technical risks.
2. The final experimental results are closely related to the effectiveness of the reagents, the related operations of the experimenter, and the experimental environment at the time. Be sure to prepare sufficient specimen backups.
3. There may be slight differences in the same product in different batches, such as detection limit, sensitivity, and color development time, etc. Please perform experiments according to the instructions in the kit, and the electronic version of the website is for reference only.
4. Only the reagents used in this kit can guarantee the detection effect. Do not mix products from other manufacturers. Only by strictly following the experimental instructions of this kit will the best test results be obtained.
 
Q & A
If the experimental results are not good, please take photos of the color development results in a timely manner, keep the used slats and unused reagents, and save them properly, then contact our company's technical support to solve the problem for you. You can also refer to the following materials:
problem
Possible Causes
solution
Standard curve deviation
Insufficient pipetting and washing
Sufficient pipetting and washing
Inaccurate pipetting
Check and correct the pipette
Low precision
Insufficient washing
Wash and soak as required
Insufficient mixing and insufficient pipetting reagents
Mix well and pipette reagents
Reuse of tips, containers and films
Use a pipettor to replace a new tip, use a new container, and cover
Inaccurate loading
Check and correct the pipette
Low OD value
Inaccurate reagent volume per well
Calibrate the pipette for precise reagent addition
Incubation time is incorrect
Guarantee sufficient incubation time
Incubation temperature is incorrect
Reagents are equilibrated to room temperature and ensure accurate incubation temperature
Enzyme label or substrate is invalid
By mixing enzyme labels and substrates, the color should appear quickly to check
No stop solution was added
Add the stop solution according to the instructions in the experimental operation steps
Out of reading time readings
Read within the reading time recommended in the instructions
Sample value
Incorrect sample storage method
Store samples properly and experiment with fresh samples
Incorrect sample collection and processing methods
Take correct sample collection and processing methods
The test substance is low in the sample
Use fresh samples and repeat the experiment
Related Products: GCK Kit Analysis Instructions

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