(D)-2-hydroxyglutarate assay kit
A technology of hydroxyglutaric acid and detection kits, which is applied in the measurement of color/spectral characteristics, material analysis by observing the influence on chemical indicators, and analysis by making materials undergo chemical reactions. Long cycle, high requirements and other issues, to achieve the effect of high-precision high-throughput inspection, strong specificity, and high precision
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Embodiment 1D2
[0023] Embodiment 1D2HG detection kit components and preparation process
[0024] A. Sample treatment solution A: 11.26mL of 71% perchloric acid and deionized water to dilute to 100mL.
[0025] B. Sample treatment solution B: made by dissolving 30.8g potassium hydroxide in 100mL deionized water.
[0026] C. Reaction buffer: 1mol / L Tris-HCl buffer solution, adjust pH to 8.0 with saturated HCl solution.
[0027] D. Reaction substrate 1: Weigh 331.7 mg of nicotinamide adenine dinucleotide (NAD) powder, dissolve it in 5 mL of deionized water, and then dilute it 10 times to obtain a 10 mmol / L NAD solution.
[0028] E. Reaction substrate 2: Weigh 14.63 mg of tetrazolium salt (MTS) powder in the dark, dissolve it in 1 mL of DPBS buffer solution, and then dilute it 10 times to obtain a 3 mmol / L MTS solution.
[0029] F. Reaction substrate 3: Weigh 2.53 mg of N-methylphenazine methosulfate (PMS) powder in the dark, dissolve it in 1 mL of DPBS buffer solution, and then dilute it 10 ti...
Embodiment 2D2
[0032] Embodiment 2D2HG detection kit detection method
[0033] (1) Reagent preparation: Reaction buffer, reaction substrate 1 / 2 / 3, enzyme, standard, sample treatment solution A / B are dissolved at room temperature, mixed separately and placed on ice for later use.
[0034] (2) Preparation of D2HG standard substance: Dilute the standard substance to two concentrations of 128 μM and 96 μM with deionized water, 400 μL each. Then the 128 μM standard was diluted in half to 64 μM, 32 μM, 16 μM, 8 μM, and the 96 μM standard was diluted in half to 48 μM to form 200 μL of 0, 8, 16, 32, 48, 64, 96, and 128 μM D2HG standards respectively. Gradient concentration solutions were kept on ice for later use.
[0035] (3) Sample treatment: 50 μL of pre-cooled sample treatment solution A was added to 200 μL of serum sample, vortexed, placed on ice for 5 min, and then centrifuged at 14,000 g at 4°C for 8 min. Take 150 μL of supernatant to a new 1.5 mL EP tube, add 8.6 μL of pre-cooled sample tr...
Embodiment 3D2
[0040] Example 3 D2HG Detection Kit Precision and Accuracy Experiment
[0041] The D2HG standard was dissolved in the reaction buffer to prepare samples with concentrations of 0, 2, 10, 20, 40, 80, 100, and 120 μM, respectively. Each sample is divided into 5 parts, measured with a D2HG detection kit, and the average value and standard deviation are calculated to obtain the precision (CV%) and recovery (%) within the batch; meanwhile, the D2HG subpackaged samples are continuously measured for 5 day, calculate the mean value, standard deviation, and obtain the inter-assay precision (CV%) and recovery (%). As shown in the table below:
[0042]
[0043]
[0044] The results showed that the D2HG detection kit had good precision, and the intra-assay and inter-assay precision CVs were both less than 5.9%. The recovery rate of the sample test ranged from 86.1% to 107.75%, indicating that the D2HG detection kit has a high accuracy.
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