Mouse 8-Hydroxydeoxyguanosine (8-OHdG) ELISA Kit This kit is for in vitro studies only
Intended application
Quantitative determination of 8-hydroxydeoxyguanosine (8-OHdG) in mouse tissue homogenate, serum, plasma, cell culture supernatant, urine or other related biological fluids by ELISA.
Overview
The C-8 position of the base guanine on the DNA chain is easily attacked by hydroxyl radicals and singlet oxygen and undergoes hydroxylation to form the adduct 8-hydroxy-2'-deoxyguanosine, 8- OHdG). 8-OHdG is a specific product of DNA oxidative damage, and is a recognized biomarker for DNA oxidative damage by endogenous and exogenous factors. 8-OHdG is one of the main products of DNA oxidative damage. During the replication process, 8-OHdG on the DNA strand can be paired with other bases other than C to form point mutations. The occurrence of GC → TA mutations has been confirmed by a large number of studies and is considered to be carcinogenic and mutagenic by oxidative stress factors. One of the main mechanisms. When the body repair mechanism is normal, 8-OHdG can be excised from the DNA strand under the action of hOGG1 and other enzymes and re-incorporated into the normal guanine base, and the excised 8-OHdG can be excreted into the bloodstream and urine. 8-OHdG exists steadily in the body. Once formed, it will not be further metabolized by the body. The content of 8-OHdG in urine is related to the degree of oxidative DNA damage in cells. At present, the body's 8-OHdG level has been widely accepted as a marker for evaluating DNA oxidative damage and is used to estimate the risk of oxidative stress-related cancer.
Experimental principle
The microtiter plate is coated with purified antibody to make a solid phase carrier, and the specimen or standard, biotinylated anti-8-OHdG antibody, and HRP-labeled affinity are added to the microwell coated with anti-8-OHdG antibody in sequence. After thorough washing, it is developed with the substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The color depth is positively correlated with 8-OHdG in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the sample concentration was calculated.
Kit composition and reagent preparation
1. ELISA plate: one piece (96 wells)
2. Standard product (lyophilized product): 2 bottles, each of which is diluted with sample diluent to 1ml before use. After being capped, it is allowed to stand for more than 10 minutes, and then repeatedly inverted / rubbed to help dissolve. Its concentration is 5,000 pg / ml, after serial dilutions (note: do not directly perform dilutions in the plate), dilute 5,000 pg / ml, 2,500 pg / ml, 1,250 pg / ml, 625 pg / ml, 312 pg / ml, 156 pg / ml, 78 pg / ml, the sample dilution is directly used as the standard concentration of 0 pg / ml, prepared within 15 minutes before use.
To prepare 2,500 pg / ml standard: Take 0.5ml (not less than 0.5ml) of the above standard at 5,000 pg / ml and add it to an Eppendorf tube containing 0.5ml of sample diluent, mix well.
3. Sample diluent: 1 × 20ml / bottle.
4. Test the diluent A: 1 × 10ml / bottle.
5. Test diluent B: 1 × 10ml / bottle.
6. Detection solution A: 1 × 120ul / bottle (1: 100), diluted with detection dilution A 1: 100 before use, prepared according to the pre-calculated total amount required for each experiment before dilution (100ul per well) , The actual preparation should be more 0.1-0.2ml. For example, 10 ul detection solution A plus 990 ul detection dilution A is prepared, mix gently, and prepare within one hour before use.
7. Test solution B: 1 × 120ul / bottle (1: 100) is diluted 1: 100 with test diluent B before use. The dilution method is the same as that of Test Solution A.
8. Substrate solution: 1 × 10ml / bottle.
9. Concentrated washing solution: 1 × 30ml / bottle, each bottle is diluted 25 times with distilled water.
10. Stop solution: 1 × 10ml / bottle (2N H2SO4).
11. Lamination: 1 × 5 sheets
Collection and preservation of specimens
1. Serum: Whole blood specimens should be left at room temperature for 2 hours or overnight at 4 ° C and centrifuged at 1000 xg for 20 minutes. Supernatant can be taken for detection, or the specimens should be stored at -20 ° C or -80 ° C, but avoid repeated Freeze and thaw.
2. Plasma: EDTA or heparin can be used as anticoagulant. Centrifuge the sample at 2-8 ° C 1000 xg for 15 minutes within 30 minutes after collection, or store the specimen at -20 ℃ or -80 ℃, but avoid repeated freezing melt.
3. Cell culture supernatant or other biological specimens: centrifuge at 1000 x g for 20 minutes, take the supernatant for testing, or store the specimen at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing.
4. Tissue homogenate: Wash the tissue samples of the animal with PBS first to remove excess blood. After homogenization, place it in 20ml PBS and place at -20 ℃ overnight. The next day, after repeated freezing and thawing, the membrane will be broken. The homogenate was centrifuged at 5000 x g for 5 minutes, and the supernatant was taken for detection.
5. Sample processing: Serum or plasma specimens are recommended to be diluted 10 times. For example: 10 times dilution, take 10ul serum or plasma and add 90ul sample diluent. It is recommended that each laboratory conduct pre-experiment before operation to establish the optimal dilution factor.
Note: The above specimens should be stored at 4 ℃ for less than 1 week, -20 ℃ or -80 ℃ should be sealed, -20 ℃ should not exceed 3 months, -80 ℃ should not exceed 6 months; specimen hemolysis will affect the final As a result of the test, hemolytic specimens should not be tested; specimens with high blood lipids do not require special treatment and can be tested directly.
Steps
Before the experiment, please prepare all the reagents in advance; when the reagents or samples are diluted, they should be mixed well, and try to avoid foaming when mixing. A standard curve should be made for each test. If the sample concentration is too high, the sample diluent should be used for dilution to make the sample meet the detection range of the kit. It is recommended that each laboratory conduct pre-experiment before operation to establish the optimal dilution factor.
1. Add sample: set blank hole, standard hole and sample hole to be tested respectively. Add 100ul of sample diluent to the blank well, and 100ul of the standard or the sample to be tested in the remaining well. Be careful not to have air bubbles. Add the sample to the bottom of the well of the microtiter plate. The target plate is covered with a cover or film and reacted at 37 ° C for 120 minutes.
To ensure the validity of the experimental results, please use a new standard solution for each experiment.
2. Discard the liquid and spin dry without washing. Add 100ul of detection solution A working solution (prepared within one hour before use) to each well, 37 ℃, 60 minutes.
3. After incubating for 60 minutes, discard the liquid in the hole, spin dry, wash the plate 3 times, soak for 1-2 minutes each time, 350ul / per hole, spin dry (you can also pat the liquid in the hole to pat dry).
4. Add 100 μl of testing solution B working solution (same as testing A working solution) to each well at 37 ℃ for 60 minutes.
5. After incubating for 60 minutes, discard the liquid in the hole, spin dry, wash the plate 5 times, soak for 1-2 minutes each time, 350ul / per hole, spin dry (you can also pat the liquid in the hole to pat dry).
6. Add 90ul of substrate solution to each well in sequence, and develop color at 37 ° C in the dark (within 30 minutes, at this time, the first 3-4 wells of the standard product have a visible blue gradient, and the latter 3-4 wells are not obvious , You can terminate).
7. Add 50ul of stop solution to each well in sequence to stop the reaction. At this time, the blue color turns to yellow. The order of adding the stop solution should be the same as that of the substrate solution. In order to ensure the accuracy of the experimental results, the termination solution should be added as soon as possible after the substrate reaction time expires.
8. Measure the optical density (OD value) of each well in sequence using an enzyme-linked instrument at a wavelength of 450 nm. Test immediately after adding stop solution.
Note:
1. Leave one hole for each experiment as a blank zero adjustment hole (different from the blank hole). No reagents are added to this hole, but the substrate solution and 2N H2SO4 are added at the end. Use this hole to adjust the OD value to zero when measuring.
2. Incubate in strict accordance with the prescribed time and temperature to ensure accurate results. All reagents must reach room temperature before use. Refrigerate the reagent immediately after use.
3. Incorrect plate washing can lead to inaccurate results. Before adding test solution B or substrate, make sure to absorb the liquid in the well as much as possible. In order to prevent the sample from evaporating, place the reaction plate in a closed box covered with a damp cloth during the test. The enzyme label plate is covered with a cover or film to avoid liquid evaporation; the next step should be performed as soon as possible after washing the plate to avoid the length of the enzyme label plate Time is dry.
4. Eliminate the residual liquid and fingerprints on the bottom of the board, otherwise it will affect the OD value.
5. Avoid direct light irradiation during storage and incubation.
6. Store unused microplates or reagents at 2-8 ° C. Standard products, test solution A working fluid, and test solution B working fluid should be configured and used according to the required amount. Please accurately configure the standard product and working fluid, and try not to configure it in a small amount (for example, when drawing test solution A, it should not be less than 10ul at a time), To avoid concentration errors due to inaccurate dilution; the detection solutions A, B, and substrate solutions should be incubated at 37 ° C for 30 minutes before use; do not reuse the diluted standard and detection solution A Working fluid or detection solution B working fluid.
7. It is recommended to set a double-hole test when testing samples to ensure the accuracy of the test results.
Washing method
Manual plate washing method: suck up (do not touch the wall) or shake off the liquid in the enzyme plate; place a few layers of water on the experimental table
Paper, with the microtiter plate down and vigorously pat several times; inject at least 0.3ml of the recommended wash buffer into the well and soak for 1-2 minutes, as needed
Yes, repeat this process several times.
Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used skillfully.
Specificity
This kit can simultaneously detect recombinant or natural mouse 8-hydroxydeoxyguanosine (8-OHdG) and has no cross-reactivity with other related proteins.
Calculation
Take the concentration of the standard as the abscissa (logarithmic coordinate) and the OD value as the ordinate (ordinary coordinate), draw a standard curve on semi-logarithmic coordinate paper (recommended to use professional curve making software for analysis, such as curve expert 1.3) , According to the OD value of the sample, find the corresponding concentration from the standard curve, and then multiply it by the dilution factor; or use the standard concentration and OD value to calculate the regression equation of the standard curve, and substitute the OD value of the sample into the equation to calculate the sample concentration , Multiplied by the dilution factor, which is the actual concentration of the sample.
Precautions
1. The washing process is very important. Insufficient washing can easily cause false positives.
2. It is best to control the sampling time within 5 minutes. If the number of specimens is large, it is recommended to use a multi-channel pipette to add samples.
3. Please make a standard curve at the same time of each measurement, and it is best to make a double hole.
4. If the content of the substance to be tested in the specimen is too high, please dilute it and then determine it. When calculating, please multiply by the dilution factor.
5. When preparing standard products and testing solution working fluids, please prepare with corresponding diluent, not to be confused.
6. Please keep the substrate away from light.
Detection range: 78 pg / ml-5,000 pg / ml
Minimum detection limit: 39 pg / ml
Explanation
1. Avoid exposing the reagent to strong light during storage and incubation. All reagent bottle caps must be tightly closed to prevent evaporation and contamination. Reagents should be protected from microbial contamination, because the interference of proteolytic enzymes will lead to erroneous results.
2. Aspirate the reagents carefully and strictly observe the given incubation time and temperature. Please note that when drawing samples / standards, enzyme conjugates or substrates, if the time interval between the first well and the last well is too large, it will result in different "pre-incubation" time, which obviously Affect the accuracy and repeatability of the measured value. Moreover, insufficient washing will affect the test results.
3. Storage of the kit: short-term (within 2 weeks) subject to the label, long-term storage Please store all reagents at -20 ℃.
4. Salt will precipitate out in the concentrated washing liquid, and it can be heated and dissolved in the water bath when diluted.
5. There may be some water-like substances in the well of the enzyme-linked plate just opened. This is a normal phenomenon and will not have any impact on the experimental results.
6. All samples should be well managed, and the samples and testing devices should be processed according to the prescribed procedures.
7. Validity: 6 months
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