Protein chip for rapid quantitative determination of bacterial fruit blotch of watermelon and preparation method thereof
A quantitative detection and protein chip technology, which is applied in measurement devices, instruments, scientific instruments, etc., to achieve the effects of simple operation, good promotion prospects, and good specificity
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Embodiment 1
[0028] Embodiment 1: as figure 1 As shown, a protein chip for rapid quantitative detection of watermelon fruit spot bacteria is a biochip in which at least one watermelon fruit spot bacteria capture antibody D is immobilized on a chip substrate. An enzyme-labeled IgG antibody is also immobilized on the substrate as a positive control P, and a spotting buffer as a negative control N; the substrate is a nitrocellulose membrane (NC). The enzyme-labeled IgG is alkaline phosphatase-labeled IgG, which is fixed on the chip base as a positive control for protein chip quality control. See figure 1 , on the substrate from left to right in the order of positive control P, negative control N, and capture antibody D, and spot 0.2 μL of each point sequentially, including 4 replicates of positive control and 4 replicates of negative control, and 8 replicates of capture antibody.
Embodiment 2
[0029] Embodiment 2: A kind of preparation method of the protein chip of rapid quantitative detection watermelon fruit spot bacterium, comprises the following steps:
[0030] a. Substrate pretreatment: the substrate needs to be treated with ddH before use 2 O soaking, and drying constant; The soaking time is at least 15min, preferably 30min; The drying temperature is 15-37°C, preferably 25°C;
[0031] b. Chip spotting preparation: Dilute the capture antibody with spotting buffer, and take enzyme-labeled IgG and spotting buffer as positive control and negative control respectively, spot and fix according to the design; the spotting buffer Liquid is 0.001-0.5mol / L, the carbonate buffer solution that pH value is 7.0-10.0, preferably 0.05mol / L, the carbonate buffer solution that pH is 9.6, and carbonate buffer solution is Na 2 CO 3 1.59 g, NaHCO 3 2.93 g, NaN 3 0.2 g, ddH 2 O 1000 mL; the concentration of the capture antibody is 20-80mg / L, preferably 80 mg / L; the described...
Embodiment 3
[0032] Embodiment 3: the preparation of watermelon fruit blotch protein chip
[0033] Nitrocellulose membrane substrate with ddH 2 Soak in O for 30 min and dry at 25°C for 30 min. Dilute the capture antibody to 80mg / L with 0.05mol / L carbonate buffer, and use alkaline phosphatase-labeled IgG as a positive control, and carbonate buffer as a negative control. See figure 1 , on the substrate from left to right in the order of positive control P, negative control N, and capture antibody D, and spot 0.2 μL of each point sequentially, including 4 replicates of positive control and 4 replicates of negative control, and 8 replicates of capture antibody. After sample application, the substrates were placed in a wet box, fixed at 37°C for 20 min, and stored at 4°C for later use. The capture antibody and detection antibody used were purchased from Agdia Company, Cat. No. SRA 14800.
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