Rat Endostatin (ES) ELISA Test Kit Instructions

Rat (Rat) Endostatin (ES) ELISA Test Kit This reagent is for research use only Specimens: serum or plasma

Test principle:
The ES kit is a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA). Standards with known ES concentrations and samples with unknown concentrations are added to microwell enzyme plates for detection. First, ES and biotin-labeled antibodies are incubated simultaneously. After washing, HRP labeled with avidin was added. After incubation and washing, the unbound enzyme conjugate is removed, and then substrates A and B are added to act simultaneously with the enzyme conjugate. Produce colors. The color depth is proportional to the concentration of ES in the sample.

Kit contents and preparation kit components (stored at 2-8 Â° C) 96-well configuration 48-well configuration Preparation
96/48 serving microplate 1 plate (96T) half plate (48T) ready-to-use plastic membrane plate cover 1 half-plate ready-to-use standard: 240pg / ml 1 bottle (0.6ml) 1 bottle (0.3 ml) Carry out dilute blank control according to the instructions 1 bottle (1.0 ml) 1 bottle (0.5 ml) ready-to-use standard dilution buffer 1 bottle (5 ml) 1 bottle (2.5 ml) ready-to-use biotin-labeled anti-ES antibody 1 bottle (6ml) 1 bottle (3.0ml) ready-to-use affinity streptavidin-HRP 1 bottle (10ml) 1 bottle (5.0ml) ready-to-use wash buffer 1 bottle (20ml) 1 bottle (10ml) according to the instructions Dilute Substrate A 1 bottle (6.0ml) 1 bottle (3.0ml) ready-to-use substrate B 1 bottle (6.0ml) 1 bottle (3.0ml) ready-to-use stop solution 1 bottle (6.0ml) 1 bottle (3.0 ml) ready to use

Bring your own materials
1. Distilled water.
2. Sampler: 5ul, 10ul, 50ul, 100ul, 200, 500ul, 1000ul.
3. Oscillator and magnetic stirrer etc.

safety
1. Avoid direct contact with the stop solution and substrates A and B. Once exposed to these liquids, rinse with water as soon as possible.
2. Do not eat, drink, smoke or use cosmetics during the experiment.
3. Do not use your mouth to absorb any ingredients in the kit.

Operation notes
1. Reagents should be stored according to the label instructions and returned to room temperature before use. The sparse standards should be discarded and cannot be stored.
2. The slats not used in the experiment should be immediately returned to the packaging bag, sealed and stored to avoid deterioration.
3. Unused other reagents should be packed or covered. Do not mix reagents of different batches. Use before warranty.
4. Use a disposable pipette tip to avoid cross-contamination, and avoid using a sampler with a metal part when drawing the stop solution and substrates A and B.
5. Use a clean plastic container to prepare the washing solution. Mix all components and samples in the kit thoroughly before use.
6. When washing the enzyme-labeled plate, it should be fully patted dry. Do not put the absorbent paper directly into the enzyme-labeled reaction well to absorb water.
7. Substrate A should evaporate to avoid opening the lid for a long time. Substrate B is sensitive to light and avoid prolonged exposure to light. Avoid contact with hands, it is toxic. The OD value should be read immediately after the experiment is completed.
8. The order of adding reagents should be the same to ensure that all wells are incubated for the same time.
9. Perform the incubation operation according to the time, the amount and order of the liquid indicated in the instructions.

Sample collection, processing and storage methods
1. Serum-Avoid any cell stimulation during the operation. Use test tubes free of pyrogens and endotoxins. After collecting blood, centrifuge at 1000 Ã— g for 10 minutes to separate the serum and red blood cells quickly and carefully.
2. Plasma ----- EDTA, citrate and heparin plasma can be used for detection. Centrifuge at 1000 Ã— g for 30 minutes to remove particles.
3. The cell supernatant --- 1000 Ã— g centrifuged for 10 minutes to remove particles and polymers.
4. Storage ------ If the sample is not used immediately, it should be divided into small parts and stored at -70 â„ƒ to avoid repeated freezing. If possible, do not use hemolysis or hyperlipidemia. If there are a large number of particles in the serum, centrifuge or filter before testing. Do not thaw at 37 Â° C or higher. Thaw at room temperature and ensure that the sample is thawed evenly and adequately.

Reagent preparation
1. Standards: Serial dilutions of standards should be prepared during the experiment and cannot be stored. Before dilution, the standard was mixed by shaking. The dilution ratio is as follows:
240 pg / ml (standard No. 6) The original concentration is added directly to 50ul without dilution.
120 pg / ml (Standard 5) 100ul of the original standard plus 100ul of standard dilution
60 pg / ml (No. 4 standard) 100ul of No. 5 standard is added with 100ul of standard diluent
30 pg / ml (No. 3 standard) 100ul of No. 4 standard is added with 100ul of standard dilution
15 pg / ml (Standard No. 2) Add 100ul of Standard No. 3 to 100ul of Standard Diluent
7.5 pg / ml (standard 1) 100ul of standard 2 is added with 100ul of standard diluent
0 pg / ml (blank control) The original concentration is directly added to 50ul without dilution.

2. Dilution of washing buffer (50 Ã—): 50-fold dilution with distilled water.

Steps
1. Before use, mix all reagents thoroughly. Don't make the liquid generate a lot of foam, so as to avoid adding a large number of bubbles during sample addition, which will cause errors in sample addition.
2. The number of slats required is determined by the number of samples to be tested plus the number of standard products. It is recommended to make multiple holes for each standard and blank hole. Each sample is determined according to its own quantity, and those that can use multiple holes can be used as much as possible.
3. Add 50ul of the diluted standard to the reaction well and 50ul of the sample to be tested in the reaction well. Immediately add 50ul of biotin-labeled antibody. Cover the membrane plate, gently shake and mix, and incubate at 37 Â° C for 1 hour.
4. Shake off the liquid in the wells, fill each well with washing liquid, shake for 30 seconds, shake off the washing liquid, pat dry with absorbent paper. Repeat this operation 3 times. If washing with a plate washer, the number of washes is increased once.
5. Add 80 ul of streptavidin-HRP to each well, gently shake and mix, and incubate at 37 Â° C for 30 minutes.
6. Shake off the liquid in the wells, fill each well with washing liquid, shake for 30 seconds, shake off the washing liquid, pat dry with absorbent paper. Repeat this operation 3 times. If washing with a plate washer, the number of washes is increased once.
7. Add 50ul of substrate A and B to each well, mix gently by shaking, and incubate at 37 Â° C for 10 minutes. Avoid light.
8. Remove the enzyme labeling plate and quickly add 50ul of stop solution. After adding the stop solution, the results should be measured immediately.
9. The OD value of each well was measured at a wavelength of 450 nm.

Recommended experimental protocol Standard concentration (pg / ml)
A 240 240 sample sample sample sample sample sample sample sample sample sample sample
B 120 120 sample sample sample sample sample sample sample sample sample sample sample
C 60 60 sample sample sample sample sample sample sample sample sample sample sample
D 30 30 sample sample sample sample sample sample sample sample sample sample sample
E 15 15 sample sample sample sample sample sample sample sample sample sample sample
F 7.5 7.5 sample sample sample sample sample sample sample sample sample sample sample
G 0 0 sample sample sample sample sample sample sample sample sample sample sample
H sample sample sample sample sample sample sample sample sample sample sample sample sample

Limitation
The results of standard No. 6 and above are non-linear, and accurate results cannot be obtained based on this standard curve.

Kit performance
1. Sensitivity: The smallest detection concentration is less than No. 1 standard. Linearity of dilution. The correlation coefficient R between the linear regression of the sample and the expected concentration is 0.990.
2. Specificity: Does not react with other cytokines.
3. Repeatability: The coefficients of variation within and between plates are less than 10%.

Judgment and analysis of results
1. Instrument value: read the OD value of each well on a microplate reader with a wavelength of 450nm

2. Taking the absorbance OD value as the ordinate (Y) and the corresponding ES standard concentration as the abscissa (X), make the corresponding curve. The ES content of the sample can be converted from the standard curve according to its OD value to calculate the corresponding concentration.

3. Range of detection value: 0-240pg / ml

4. Sensitivity: 0.1 pg / ml
Chinese manual UNIONHONEST
Enzyme Linked Immunosorbent Assay

Rat (Rat) Endostatin (ES) ELISA Test Kit This reagent is for research use only Specimens: serum or plasma

Bring your own materials
1. Distilled water.
2. Sampler: 5ul, 10ul, 50ul, 100ul, 200, 500ul, 1000ul.
3. Oscillator and magnetic stirrer etc.

2. Dilution of washing buffer (50 Ã—): 50-fold dilution with distilled water.

Limitation
The results of standard No. 6 and above are non-linear, and accurate results cannot be obtained based on this standard curve.

Judgment and analysis of results
1. Instrument value: read the OD value of each well on a microplate reader with a wavelength of 450nm

3. Range of detection value: 0-240pg / ml

4. Sensitivity: 0.1 pg / ml

Manual burr grinders are turned by hand, rotating one grinding surface against the other. Coffee mills usually have a handle, providing leverage for the many turns required to grind enough coffee for a cup. The ground coffee is collected in a container which is part of the mill.

Salt, pepper, and spice mills, essentially the same as coffee mills, usually do not have a handle, the entire top rotating instead. While this is less convenient, only a few turns are required to grind enough. The ground product falls directly onto the food being seasoned; the mill has no container. A few designs have abrasive surfaces which do not rotate; each squeeze of the handles moves one flat plate past another, then the plates are restored to their original position by a spring. Many hard spices are available in containers incorporating a simple cone burr grinder, intended to be discarded when empty.

Most grinders can be adjusted to set the fineness of grind.

Manual mills can be used for grinding other food products than they are intended for, but mills designed for pepper grinding are inappropriate for producing finely-ground flour. Laura Ingalls Wilder's novel The Long Winter describes a family grinding wheat in a coffee mill to make flour during months of hardship.

The first Coffee Grinder was made by Richard Dearmann, an English blacksmith from Birmingham, in 1799. Then this grinder was widely distributed in the US, where Increase Wilson patented the first wall coffee grinder in 1818.^{[citation needed]}

Peugeot of France patented a Pepper Grinder in 1842. The mechanism of case-hardened steel cracked the peppercorns before the actual grinding process. The grooves on the Peugeot mechanism were individually cut into the metal and then case-hardened, making them very durable.^{[citation needed]}

Pepper and Salt Mill

Pepper Mill, Salt Mill, Pepper Grinder, Plastic Kitchen Gadget, Kitchen Gadget

HOMEARTS INDUSTRIAL CO.,LTD , http://www.homeartschina.com