Introduction
Cytokine-induced neutrophil chemoattractant factor 1 (CINC-1) was originally purified by interleukin 1β stimulation of rat renal epithelial cells (NRK-52E). In 1989, the Watanabe team of Toyama Medical and Pharmaceutical University identified The amino acid sequence of rat CINC-1 is shown. CINC-1 is a member of the CXC chemokine subfamily. Several other rat CXC chemokines (CINC-2α, CINC-2β, CINC-3 / MIP- 2) The sequence has also been identified, about 63-67% of the amino acid sequence is the same as CINC-1. In addition, GROα, GROβ and GROγ are also 68%, 71%, 69% and CINC-1 sequence respectively. This indicates that CINCs are rat analogs of human GROs.
principle
This kit uses two specific antibodies. After washing the plate, TMB substrate solution is added for color development.The intensity of color development is proportional to the amount of rat GRO / CINC-1 in the sample.
examination range
4.69 ~ 300 pg / mL
expected usage
â– This IBL kit can be used for quantitative detection of GRO / CINC-1 in rat serum, EDTA plasma, and cell supernatant
â– Both recombinant and natural rat GRO / CINC-1 can be detected.
Kit components
1 Pre-coated plate: anti-rat GRO / CINC-1 rabbit IgG, affinity purified 96T
2 Enzyme labeled antibody:
(30-fold concentration) HRP labeled anti-rat GRO / CINC-1 rabbit IgG, affinity purified 0.4mL x 1
3 Standard: Recombinant rat GRO / CINC-1 0.5mL x 2
4 EIA buffer: 1% BSA, 0.05% Tween 20 BPS 30mL x 1
5 Labeled antibody dilution: 1% BSA, 0.05% Tween 20 BPS 12mL x 1
6 Developer: TMB substrate solution 15mL x 1
7 Stop solution: 1N sulfuric acid 12mL x 1
8 Concentrated washing solution:
(40 times concentrated) with 1% BSA, 0.05% Tween 20 BPS 50mL x 1
Instructions
1 The equipment needed for the experiment (but the kit is not provided)
Microplate reader (450nm) micro pipette and its nozzle
Distilled water in measuring cylinder and beaker
Refrigerator (4 ° C) Coordinate paper (log / log)
Absorbent paper test tube (for standard dilution)
Incubator (37 ° C ± 1 ° C)
Wash bottles (for washing plates)
Disposable reagent tubes (for concentrated enzyme-labeled antibodies and color reagents)
2 Preparation
Preparation of washing solution
The washing solution of the kit is a 40-fold concentrated solution. Dilute 50 mL of concentrated washing solution with 1950 mL of distilled water to make 2000 mL of ready-to-use diluted washing solution. Store in the refrigerator for 2 weeks.
Preparation of enzyme-labeled antibodies
The labeled antibody in the kit is a 30-fold concentrate, which is diluted 30-fold with the labeled antibody dilution according to the required amount.
Example:
If you use one plate (8 wells), you need 800 μL of labeled antibody. Just dilute 30 μL of antibody concentrate with 870 μL of labeled antibody diluent and mix well. Add 100 μL to each well
Concentrated enzyme-labeled antibodies need to be diluted before use in experiments
The remaining diluted labeled antibody solution should be sealed and stored at 4 ° C
Standard preparation
Add 0.5 mL of distilled water to the bottled standard to make a rat GRO / CINC-1 standard solution with a concentration of 600 pg / mL
Standard dilution
Prepare 8 test tubes and add 230 μL of EIA buffer to each tube. Dilute to the following concentration, as shown in the figure below:
Test tube 1 300 pg / mL
Test tube 2 150 pg / mL
Test tube 3 75 pg / mL
Test tube 4 37.5 pg / mL
Test tube 5 18.75 pg / mL
Test tube 6 9.38 pg / mL
Test tube 7 4.69pg / mL
Test tube 80 pg / mL (sample blank control)
Pipette 230ul of the standard solution into the No. 1 tube and mix it. Then draw 230ul from the No. 1 tube and add it to the No. 2 tube to mix as shown in the figure below.
Sample dilution
If the sample needs to be diluted, it must be diluted with EIA buffer
If the concentration range of rat GRO / CINC-1 in the sample is unknown, prepare several sample solutions with different dilution gradients in advance to determine the optimal detection concentration.
3 Experimental steps
Before use, all reagents should be equilibrated to room temperature, about 30min, and mixed thoroughly to ensure that the quality of the reagents does not change. The standard curve and sample detection must be performed simultaneously.
Reagent to be tested 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 and incubate at 37 ° C for 1 hour
Wash the plate 7 times
Enzyme labeled antibody 100ul 100ul 100ul-
Cover plate and incubate at 37 ° C for 30 min
Wash plate 9 times
Developer 100ul 100ul 100ul 100ul
Incubate at room temperature in the dark for 30min
Stopping fluid 100ul 100ul 100ul 100ul
After adding the stop solution, read the OD value at 450nm within 30min
1) Set reagent blank control and add 100ul EIA buffer to the corresponding microwell
2) Determine the sample blank control well, test the sample well and the standard well, add 100ul of the sample control (tube 8), the test sample and the diluted standard (tube 1-7) to the corresponding microwells in.
3) Cover the plate and incubate at 37 ° C for 1 hour
4) Wash the plate vigorously with a washing bottle, then add diluted washing solution, let stand for 15-30 seconds, and then pour the reaction solution in the well thoroughly
Repeat plate washing 7 times.
Finally pat dry on absorbent paper to remove residual droplets
If you use an automatic plate washer to wash the plate, first wash it automatically 4 times, and then wash it manually 3 times with the wash bottle as above
5) In addition to the reagent blank control wells, add 100ul of enzyme-labeled antibody to each well
6) Cover plate, incubate at 37 ° C for 30min
7) Follow the above steps 4) Wash the plate 9 times
8) Use a disposable test tube to take the required amount of developer, and add 100ul of each developer to the microwells. To avoid contamination, do not pour the remaining reagents back into the reagent bottles
9) Incubate at room temperature in the dark for 30 minutes. After adding the developer, the color of the solution will turn blue
10) Add 100ul of stop solution to each well, the color of the solution will turn yellow
11) Wipe off the stains or water droplets on the bottom of the microwell plate to ensure that no bubbles are generated in the solution in the microwells. Read the results at 450nm of the microplate reader within 30min after adding the stop solution
pay attention
Test the samples as soon as they are collected. If you need to store them, freeze the samples, but avoid repeated freezing and thawing. Before testing, thaw the samples to room temperature.
If the sample needs to be diluted, dilute with EIA buffer
It is recommended that samples and standards be tested in duplicate
Keep the sample in the neutral pH range. Organic solvent contamination will affect the experimental results
Only wash the plates with the washing solution provided by this kit. Insufficient plate washing will cause erroneous results
When patting dry on absorbent paper to remove residual droplets, do not scratch the inner wall of the hole with absorbent paper
The display agent should be kept away from light and avoid contact with metal substances
After adding the stop solution, the experimental results should be read within 30 minutes
Result calculation
All experimental data (including the OD value of the standard and the test sample) should be subtracted from the OD value of the sample blank control.
For log-log graph paper, use the corrected OD value as the ordinate and the corresponding concentration as the abscissa to establish a standard curve. The samples directly obtain the concentration value on the standard curve.
Standard curve demonstration
The typical standard curve shown in the above figure is only for demonstration and cannot be used to obtain experimental data. The standard curve should be established for each experiment.
General matters
1 Store all reagents at 2-8 ° C. Allow the reagents to equilibrate to room temperature (about 30 minutes) before the experiment
2 The bottled standard product is a lyophilized product, so carefully open the cap
3 The stop solution is a strong acid substance. When using, avoid contact with the skin and clothing.
4 Rinse with water before disposing of used materials
5 Concentrated enzyme-labeled antibody, EIA buffer and concentrated washing solution may precipitate, but this does not affect the experiment
6 Wash hands after using reagents
7 Do not mix reagents with different batches
8 Do not use expired reagents
9 The kit is for research use only and cannot be used for clinical diagnosis.
Storage and expiration date
Storage conditions: 2-8 ° C
The expiration date is shown in the outer packaging of the kit
This translation is for reference only, please refer to the original text for details.
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