Human nuclear factor κB subunit p65 affinity peptide (NF-κB p65) ELISA kit

Human nuclear factor κB subunit p65 affinity peptide (NF-κB p65) ELISA kit is for in vitro research use only, not for clinical diagnosis!

Detection range: 1.56 ng / ml-100 ng / ml, please use the following concentration values ​​for drawing standard curve: 100 ng / ml, 50 ng / ml, 25 ng / ml, 12.5 ng / ml, 6.25 ng / ml, 3.12 ng / ml, 1.56 ng / ml.

Minimum detection limit: 0.39 ng / ml

Experimental principle: The microplate is coated with purified NF-κB p65 antibody to make a solid-phase carrier. Samples or standards, biotinylated NF-κB p65 antibody, and HRP-labeled avidin are added to the microwells in sequence. After thorough washing, develop color with 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 NF-κB p65 in the sample. The absorbance (value) was measured with a microplate reader at a wavelength of 450nm, and the sample concentration was calculated.

Specificity: This kit can detect recombinant or natural human NF-κB p65 at the same time, and does not cross-react with other related proteins.

Intended application: ELISA method for quantitative determination of NF-κB p65 in human serum, plasma, tissue homogenate or other related biological fluids.

Composition and preparation of ELISA kit for human nuclear factor κB subunit p65 affinity peptide (NF-κB p65)

1. Enzyme plate: one piece (96 wells)

2. Standard product (lyophilized product): 2 bottles, please prepare within 15 minutes before use. Each bottle is diluted to 1ml with sample diluent. After capping, it is allowed to stand at room temperature for about 10 minutes. At the same time, it is inverted / rubbed repeatedly to help dissolve. Its concentration is 500 ng / ml. After dilution to 100 ng / ml, do Serial multiple dilutions (Note: Do not directly perform multiple dilutions in the plate), respectively formulated into 100 ng / ml, 50 ng / ml, 25 ng / ml, 12.5 ng / ml, 6.25 ng / ml, 3.12 ng / ml , 1.56 ng / ml, the sample dilution is directly used as a blank well 0 ng / ml. For example, to prepare a 50 ng / ml standard: take 0.5 ml (not less than 0.5 ml) of 100 ng / ml of the above standard and add it to an Eppendorf tube containing 0.5 ml of sample diluent, mix well, and so on for the remaining concentrations.

3. Sample dilution: 1 × 20ml.

4. Test dilution A: 1 × 10ml.

5. Test dilution B: 1 × 10ml.

6. Detection solution A: 1 × 120 per bottle (1: 100). Before use, dilute with Test Diluent A 1: 100 (for example: 10 Test Solution A / 990 Test Diluent A), mix well, and prepare according to the pre-calculated total amount required for each experiment (100 / Hole), more 0.1-0.2ml should be prepared during actual preparation.

7. Detection solution B: 1 × 120 per bottle (1: 100). Dilute 1: 100 with test dilution B immediately 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 × 10 ml / bottle (2 mol / L H2SO4).

11. Lamination: 5 sheets

12. Instruction manual: 1 copy

Bring your own items:

1. Distilled or deionized water, filter paper

2. Microplate reader (it is recommended to preheat the instrument before use)

3. Micro-liquid dispenser and pipette tip, EP tube

Collection and storage of specimens:

1. Plasma: EDTA or heparin can be used as an anticoagulant. Centrifuge the sample at 1000 g for 15 minutes within 30 minutes after collection. The supernatant can be taken for detection, or the supernatant should be stored at -20 or -80, but avoid repeated Freeze and thaw.

2. Serum: Whole blood samples should be left at room temperature for 2 hours or 4 overnight. After centrifugation at 1000 g for 20 minutes, take the supernatant for detection, or store the supernatant at -20 or -80, but avoid repeated freeze-thaw cycles .

3. Tissue homogenate: Wash the tissue samples of the animal with PBS first to remove excess blood. After homogenization, put it in 5 ~ 10 ml PBS and place at -20 ℃ overnight. The next day, after repeated freezing and thawing The membrane was centrifuged at 5000x g for 5 minutes, and the supernatant was taken for detection.

4. Other biological specimens: centrifuge at 1000 g for 20 minutes, take the supernatant for testing, or store the supernatant at -20 or -80, but avoid repeated freezing and thawing.

Note: The above specimens should be sealed and stored for less than 1 week, -20 should not exceed 1 month, -80 should not exceed 2 months; specimen hemolysis will affect the final test results, so hemolysis specimens should not be tested.

Steps:

Before starting the experiment, all reagents should be equilibrated to room temperature, and the reagents should not be dissolved directly at 37; when preparing reagents or samples, they must be thoroughly mixed, and try to avoid foaming when mixing. The sample content should be predicted before the experiment. If the sample concentration is too high, the sample should be diluted to make the diluted sample meet the detection range of the kit, and then multiplied by the corresponding dilution factor when calculating.

1. Add sample: set blank hole, standard hole and sample hole to be tested respectively. Add sample diluent 100 to blank wells, and standard or sample 100 to be tested in the remaining wells. Be careful not to have air bubbles. Add the sample to the bottom of the microtiter plate when adding samples. Try not to touch the walls of the wells. Cover or cover the target plate and incubate at 37 for 2 hours. To ensure that the experimental results are valid

For each experiment, please use a new standard solution.

2. Discard the liquid and spin dry without washing. Add 100 working solutions of detection solution A (prepared before use) to each well, add the microplate to the membrane, and incubate at 37 for 1 hour.

3. Discard the liquid in the hole, spin dry, wash the plate 3 times, soak for 1-2 minutes each time, about 400 / per hole, spin dry (you can also pat dry the liquid in the hole).

4. Add 100 working solution of detection solution B (prepared before use) to each well, add a film, and incubate at 37 for 1 hour.

5. Discard the liquid in the hole, spin dry, wash the plate 5 times, the method is the same as step 3.

6. Add substrate solution 90 per well, enzyme label plate and film 37 to avoid color development (reaction time is controlled at 15-30 minutes, when the first 3-4 wells of the standard wells have a clear blue gradient, the last 3 -When the gradient of 4 wells is not obvious, it can be terminated).

7. Add stop solution 50 to each well 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.

8. Immediately measure the optical density (value) of each well with a microplate reader at a wavelength of 450nm.

Note:

1. Reagent preparation: All reagents should be equilibrated to room temperature before use. Please store the reagents according to the instructions immediately after use. Please use disposable tips during the experiment to avoid cross contamination.

2. Add sample: When adding sample or adding reagent, if the time interval between the first well and the last well is too large, it will result in different "pre-incubation" time, which will obviously affect the measured value. Accuracy and repeatability. The time of one sample addition (including standard and all samples) is best controlled within 10 minutes. It is recommended to set up multiple holes for experiments.

3. Incubation: In order to prevent the sample from evaporating, place the enzyme-labeled plate with a cover or film in the wet box during the experiment to avoid liquid evaporation; the next step should be carried out as soon as possible after washing the plate, and should be avoided at any time The microplate is in a dry state; at the same time, the given incubation time and temperature should be strictly observed.

4. Washing: The washing liquid remaining in the reaction well during the washing process should be patted dry on the filter paper. Do not put the filter paper directly into the reaction well to absorb water. At the same time, the remaining liquid and fingerprints on the bottom of the plate should be eliminated to avoid affecting the final enzyme label. Instrument reading.

5. Reagent preparation: Before using, please shake your hands a few times or centrifugation to prevent the liquid on the tube wall or bottle cap from depositing to the bottom of the tube. Standard products, working solution A, and working solution B should be prepared according to the required amount, and prepared with the corresponding diluent, not to be confused. Please accurately prepare the standard and working solution, and try not to prepare in small amounts (such as when drawing the test solution A, not less than 10 at a time) to avoid concentration errors due to inaccurate dilution; do not reuse the diluted standard and test Solution A working fluid, detection solution B working fluid.

6. Control of reaction time: Please observe the color change of the reaction well regularly after adding the substrate (for example, every 10 minutes), if the color is darker, please add the stop solution in advance to stop the reaction, to avoid the reaction is too strong and affect the microplate reader Optical density reading.

7. Substrate: Please keep the substrate away from light, and avoid direct exposure to strong light during storage and incubation.

Washing method:

1. Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used skillfully.

2. Manual plate washing method: Inject at least 0.4ml of the recommended washing buffer into the well, soak for 1-2 minutes, aspirate (do not touch the plate wall) or shake off the liquid in the enzyme plate, and lay a few layers on the experimental table With absorbent paper, the microtiter plate is vigorously patted down several times; repeat this process several times as needed.

Calculation: The value of each standard and sample is deducted from the blank hole value (7-point diagram). If multiple holes are set, the average value should be used for calculation. Taking the concentration of the standard product as the ordinate (logarithmic coordinate) and the value as the abscissa (logarithmic coordinate), draw a standard curve on the logarithmic coordinate paper. It is recommended to use professional curve making software for analysis, such as curve expert 1.3, according to the sample value, find the corresponding concentration from the standard curve, and multiply it by the dilution factor; or use the concentration and value of the standard to calculate the regression equation of the standard curve, and then the sample The value of is substituted into the equation, the sample concentration is calculated, and then multiplied by the dilution factor, which is the actual concentration of the sample.

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. Preservation of the kit: some reagents are stored at -20 ℃, and some reagents are stored at 2-8 ℃, depending on the label.

4. Salt will be precipitated from the concentrated washing liquid, which can be heated and dissolved in the water bath when diluted.

5. The well of the enzyme-linked plate just opened may contain a little water-like substance. This is a normal phenomenon and will not have any impact on the experimental results.

6. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions.

7. All samples should be managed, and the samples and testing devices should be processed according to the prescribed procedures.

8. Validity: 6 months