A phosphorylated antibody chip kit for detecting human receptor tyrosine kinase
The technology of tyrosine kinase and antibody chip is applied in the field of phosphorylated antibody chip kit and phosphorylated antibody chip kit of human receptor tyrosine kinase, which can solve the problems of time-consuming, laborious, poor effect and high cost, Achieve the effect of overcoming cumbersome operation, less sample dosage and improving sensitivity
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Embodiment 1
[0033] Example 1: Screening of the best antibody chip carrier.
[0034] Conventional antibody chips mostly use glass slides as carriers. Because glass slides are fragile and the detection equipment is expensive, they are not suitable for small-scale applications, and it also brings difficulties to the transportation of reagents in the diagnostic field. However, the traditional nitrocellulose membrane is fragile and has a high background, resulting in large fluctuations in results. We screened membranes and glass slides that are not treated with active methods on the market. After a lot of screening, we found that whether it is a membrane or a glass slide, the temperature and the active reagent components on the surface are the key to the effect of spotting.
[0035] (1) Screening of diaphragm
[0036] In order to immobilize the antibody on the surface of the membrane, we screened different methods to treat the membrane, as shown in the table below. Then use a fully automatic...
Embodiment 2
[0058] Example 2: Preparation of the kit.
[0059] The antibody chip kit of the present invention comprises the following components:
[0060] Solid phase carrier: standard membrane or glass slide coated with antibody.
[0061] Wash solution: 20X concentrated wash solution containing Tween 20. 1X washing solution is pH 7.2, containing 0.1% Tween 20, 0.1mol / L phosphate buffer.
[0062] Diluent: 2 vials of 15ml 5X Concentrated Diluent D for sample dilution, 1 vial of 15ml 5X Concentrated Diluent B for antibody and HRP-streptavidin. 1X dilution B is 15mM, PBS buffer solution of pH7.4, solute and its mass concentration or molar concentration or volume concentration in said dilution B are as follows: 0.5% casein, 2-4% sucrose, 150mM NaCl . The 1X dilution D is 15mM, pH6.5 PBS buffer solution, the solute and its mass concentration or molar concentration or volume concentration in the sample dilution solution are as follows: 2-4% sucrose, 150mM NaCl.
[0063] Detection Antibody:...
Embodiment 3
[0081] Embodiment 3: Detect RTK phosphorylation protein with the kit of embodiment 2
[0082] The bottom film is placed in the matching square box. Since there are a plurality of chip lattices distributed on the bottom film of this embodiment, the square box is provided with 8 square grids in this embodiment. Each chip lattice is divided into mutually independent reaction areas. In this embodiment, 8 square boxes are arranged, and 2 ml of blocking solution is added to each square, and then incubated at room temperature for 30 minutes, and then carried out sequentially. Follow the steps below:
[0083] 1. Add sample
[0084] Aspirate the blocking solution in each square, put 100 microliters to 5 ml of the sample diluted with the blocking solution into the square with a membrane, and then shake it on a shaker at room temperature for 1 to 2 hours, or in React at 4°C for 12-18 hours.
[0085] 2. Membrane washing
[0086] Washing solution I cleaning: Aspirate the sample from th...
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