Method for detecting protein kinase based on surface plasmon resonance
A surface plasmon and protein kinase technology, applied in the chemical field, can solve the problems of complex preparation process, high cost, and poor protein non-specific ability, and achieve the effect of simple preparation, low cost, and prevention of non-specific adsorption
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Embodiment 1
[0016] The preparation of SPR sensor chip, the steps are as follows:
[0017](1) The gold film with a thickness of 50 nm was purchased from Biosensing Instrument Company, USA. The basic composition of the company's gold film is to coat a 18 mm × 18 mm glass slide with a 2 nm thick Cr metal film to enhance the bonding force between gold and glass, and then coat it with a 50 nm thick Cr metal film. Gold as the sensing layer;
[0018] (2) The gold film was soaked in absolute ethanol for 10 minutes before use, ultrasonicated for 5 minutes, rinsed with secondary water, and cleaned with N 2 blow dry;
[0019] (3) After cleaning, use H 2 The flame is annealed on the surface of the gold sheet, preheated first, and then at an angle of 45 degrees H 2 Flame uniform annealing for 30 seconds to clean the surface and eliminate non-specific adsorption;
[0020] (4) Polypeptide P 1 Dissolve to 1 mM with secondary water, then dilute to 10 µM with 10 mM pH 7.2 PBS solution, peptide P 2 D...
Embodiment 2
[0024] The specific and non-specific effects of protein kinase A on the surface of the gold film in Example 1, the steps are as follows:
[0025] (1) Protein kinase A was diluted with 10 mM pH 7.2 PBS buffer;
[0026] (2) The prepared P 1 / P 2 The modified gold membrane was placed on the SPR instrument, and when the baseline was stable, 25 nM protein kinase A solution was flow-injected, and the SPR response signal was recorded until it stabilized. The carrier liquid is 10 mM PBS buffer solution, pH 7.2, and the flow rate is 10 µL / min;
[0027] (3) The prepared P 2 The modified gold membrane was placed on the SPR instrument, and when the baseline was stable, 25 nM protein kinase A solution was flow-injected, and the SPR response signal was recorded until it stabilized. The carrier liquid is 10 mM PBS buffer solution, pH 7.2, and the flow rate is 10 µL / min;
[0028] (4) Compare the SPR response signals of the protein kinase A solution injected on the surface of different go...
Embodiment 3
[0029] Embodiment 3: the regeneration of sensor chip, the steps are as follows:
[0030] (1) Dilute protein kinase A with 10 mM, pH 7.2 PBS buffer;
[0031] (2) The prepared P 1 / P 2 The modified gold membrane was placed on the SPR instrument, and when the baseline was stable, 25 nM protein kinase A solution was flow-injected, and the SPR response signal was recorded until it stabilized. The carrier liquid is 10 mM PBS buffer solution, pH 7.2, and the flow rate is 10 µL / min;
[0032] (3) Inject 50 mM NaOH solution into the detection channel of step (2) at a flow rate of 10 µL / min to elute the protein kinase A bound to the surface of the sensing gold membrane, allowing the sensing gold membrane to regenerate until the SPR signal Reach the size before the injection of protein kinase A to prepare for the detection of the next sample;
[0033] (4) Inject the same concentration of protein kinase A solution as in step (2) onto the surface obtained in step (3), record the SPR sig...
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