Human soluble α-klotho protein detection kit
Item No.: 27998
Introduction Introduction α–klotho is a negative regulatory gene in genetically modified (similar to human aging symptoms). Its gene sequence exists in multiple species, including humans based on mouse studies. α–klotho protein is a kind of A transmembrane protein with a molecular weight of approximately 130 kDa expressed in the kidney and parathyroid glands.
In recent years, it has been confirmed that α-klotho is an important factor regulating the metabolism of minerals, such as calcium and phosphorus metabolism. Therefore, it is believed that the aging symptoms of mice are caused by the destruction of minerals due to the loss of α-klotho. At the same time, it has been reported that the long N-terminal extracellular domain that constitutes the major part of the α-klotho protein sequence can be released and released into the blood. However, there are still many doubts about the functions and changes of soluble α-klotho protein, so it is urgent to develop a set. –-klotho detection system This ELISA kit can be used to detect alpha-klotho protein in human blood.
Experimental principle <br>This solid phase ELISA kit is based on the principle of sandwich method, using two different highly specific antibodies, TMB as a substrate display agent, the color rendering intensity is proportional to the amount of human soluble α-klotho
examination range
93.75~6000pg/mL
Intended use <br>This kit can be used for the quantitative detection of human soluble α–klotho in serum, plasma and urine.
Kit composition
In recent years, it has been confirmed that α-klotho is an important factor regulating the metabolism of minerals, such as calcium and phosphorus metabolism. Therefore, it is believed that the aging symptoms of mice are caused by the destruction of minerals due to the loss of α-klotho. At the same time, it has been reported that the long N-terminal extracellular domain that constitutes the major part of the α-klotho protein sequence can be released and released into the blood. However, there are still many doubts about the functions and changes of soluble α-klotho protein, so it is urgent to develop a set. –-klotho detection system This ELISA kit can be used to detect alpha-klotho protein in human blood.
Experimental principle <br>This solid phase ELISA kit is based on the principle of sandwich method, using two different highly specific antibodies, TMB as a substrate display agent, the color rendering intensity is proportional to the amount of human soluble α-klotho
examination range
93.75~6000pg/mL
Intended use <br>This kit can be used for the quantitative detection of human soluble α–klotho in serum, plasma and urine.
Kit composition
1. Coated plate: coated with anti-human soluble α-klotho (67G3) mouse IgG monoclonal antibody, affinity purification 96T x 1
2. Concentrated labeled antibody: (30X) HRP-labeled anti-human soluble α-klotho mouse IgG Fab' affinity purification 0.4ml x1
3. Standard: Recombinant human soluble α–klotho 0.5mlx2
4. EIA buffer: 30mlx1
5. Labeled antibody dilution: 1% BSA, PBS 12mlx1 with 0.05% Tween 20
6, color developer: TMB substrate liquid 15mlx1
7, stop solution: 1N sulfuric acid 12mlx1
8. Concentrated Wash Buffer: (40X) Phosphate Buffer with 0.05% Tween 20 50mlx1
Materials required for the experiment ( but not provided in the kit )
Microplate reader (450nm) Micropipette and sampler tip cylinder and beaker Deionized water absorbent paper Coordinate paper standard dilution tube Wash disposable tube
Reagent preparation
1. Preparation of washing buffer:
First, the (40X) washing concentrate is equilibrated to room temperature, thoroughly mixed, and 50 ml of the concentrated solution is mixed with 1950 ml of deionized water to obtain. The diluted washing solution should be stored in the refrigerator and used up within 2 weeks.
2. Preparation of enzyme-labeled antibodies:
Dilute the labeled antibody to 30-fold with the labeled antibody dilution. For example, if only one plate is used, 8 wells, you need to label the antibody 800 ul (30 ul of labeled antibody + 870 ul of labeled antibody dilution, mix, use each Holes are added 100ul)
This step should be done before the labeled antibody is used. The remaining labeled antibody concentration should be stored in a sealed bottle and stored at 4 ° C.
This step should be done before the labeled antibody is used. The remaining labeled antibody concentration should be stored in a sealed bottle and stored at 4 ° C.
3. Preparation of standard products:
Pipette 0.5ml of deionized water into the standard bottle and mix well to obtain 12000pg/ml human soluble α-klotho standard.
4. Dilution of standard products:
Prepare 8 tubes for standard dilution, add 230 ul of EIA buffer to each of the following 8 tube concentrations
No. 1 tube 6000pg/ml
Tube 2 3000pg/ml
Tube No. 3 1500pg/ml
Tube 4 750pg/ml
No. 5 tube 375pg/ml
No. 6 tube 187.5pg/ml
Tube No. 7 93.75pg/ml
Tube 8 0pg/ml (test sample blank)
Pipette 230 ul of standard product into No. 1 tube and mix, then pipette 230 ul from No. 1 tube and add No. 2 tube to mix. As shown in the figure below, dilute according to this rule. The standard range is 6000~93.75pg/ml. As a sample blank, the concentration is 0pg/ml.
No. 1 tube 6000pg/ml
Tube 2 3000pg/ml
Tube No. 3 1500pg/ml
Tube 4 750pg/ml
No. 5 tube 375pg/ml
No. 6 tube 187.5pg/ml
Tube No. 7 93.75pg/ml
Tube 8 0pg/ml (test sample blank)
Pipette 230 ul of standard product into No. 1 tube and mix, then pipette 230 ul from No. 1 tube and add No. 2 tube to mix. As shown in the figure below, dilute according to this rule. The standard range is 6000~93.75pg/ml. As a sample blank, the concentration is 0pg/ml.
5. Sample dilution:
Diluted samples with EIA buffer as required. Serum, plasma or urine samples are recommended to be diluted 2 to 4 times
Experimental Procedures <br>Before use, all reagents should be equilibrated to room temperature for about 30 minutes, thoroughly mixed to ensure no change in reagent quality. Standard curve and sample detection are performed simultaneously.
Experimental Procedures <br>Before use, all reagents should be equilibrated to room temperature for about 30 minutes, thoroughly mixed to ensure no change in reagent quality. Standard curve and sample detection are performed simultaneously.
Reagent | Test sample | Standard | Sample blank | Reagent blank |
Test sample 100ul | Dilution standard (1-7 tubes) 100ul | EIA buffer (8th tube) 100ul | EIA buffer 100ul | |
Cover plate, incubated for 60 min at room temperature | ||||
Wash the plate 7 times | ||||
Enzyme-labeled antibody | 100ul | 100ul | 100ul | - |
Cover plate, incubated for 30 min at room temperature | ||||
Wash the plate 9 times | ||||
Reagent | 100ul | 100ul | 100ul | 100ul |
Incubate at room temperature for 30 min in the dark | ||||
Stop solution | 100ul | 100ul | 100ul | 100ul |
Add stop solution, read OD value at 450nm within 30min | ||||
- Set a reagent blank and add 100 ul of EIA buffer to the corresponding wells.
- Determine the sample blank control well, test the sample well and standard well, and add 100 ul of the sample control, test sample and diluted standard to the corresponding microwell.
- Cover plate, incubate for 60 min at room temperature
- Wash the plate with a washing bottle, add a washing solution to the micropores for 15-30 seconds, and discard the washing solution. Repeat 7 times, and finally pat dry on absorbent paper. If you wash the plate 4 times with a washer, you need to wash the plate 3 times with a wash bottle.
- In addition to the reagent blank control well, add 100 ul of enzyme-labeled antibody to each well.
- Cover plate, incubate for 30 min at room temperature
- Wash the plate 9 times according to the above step 4)
- Use a disposable tube to take the required amount of developer, add 100ul of each developer to the microwell. To avoid contamination, do not refill the reagent in the reagent bottle.
- Incubate at room temperature for 30 min in the dark, and the color of the solution will turn blue after adding the developer.
- Add 100 ul of stop solution to each well and the color of the solution will turn yellow.
- Wipe off the stains or water droplets on the bottom of the microplate to ensure that the solution in the micropore is free of air bubbles. Read the 450 nm at the microplate reader within 30 min after adding the stop solution.
pay attention
1. Test samples should be tested or frozen immediately after collection to avoid repeated freeze-thaw samples. Before the test, they should be completely dissolved and mixed at low temperature, especially urine samples. Temperature and freeze-thaw ratio are more stable than plasma or serum. The effect is greater, so the sample should be frozen immediately after collection to avoid repeated freezing
2. The test sample is diluted with EIA buffer as needed.
3. It is recommended to double test each test sample and standard product.
4. The test sample should be in the neutral pH range. The contamination of organic solvents may affect the experimental results.
5. Only use the washing liquid provided by this kit to wash the plate. If the washing is insufficient, the test will fail.
6, pat the microplate with absorbent paper to avoid scratching
7, TMB substrate liquid should be stored in the dark, because it is sensitive to light, and avoid contact with metal
8. After adding the stop solution, it must be read within 30 minutes.
Calculation of results <br>Before drawing the standard curve, all data (including standard and unknown samples) should be subtracted from the blank value of the test sample. The standard curve is drawn on the coordinate paper with the concentration of the standard and the corresponding OD value. The concentration of the unknown sample can be directly read from the standard curve
This standard curve is for demonstration purposes only and should not be used to obtain test data. A standard curve should be established for each test.
This translation is for reference only, please refer to the original for details.
This standard curve is for demonstration purposes only and should not be used to obtain test data. A standard curve should be established for each test.
This translation is for reference only, please refer to the original for details.
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