Protein analysis method based on hydrophobic amino acid specific protease
A technology of hydrophobic amino acid and analysis method, which is applied in the field of protein analysis based on hydrophobic amino acid-specific protease, and achieves the effect of improving the accuracy and application range, and increasing the number of identifications
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Embodiment 1
[0016] Short-chain hydrophobic amino acid-specific protease for the analysis of myoglobin
[0017] (1) Take 0.2mg of Myoglobin and dissolve it in 200μL of 50mM Tris-HCl, 8M Urea, 150mMNaCl, 20mM CaCl 2 In the solution, the pH is 5.0, so that the concentration of Myoglobin in the solution is 1 mg / mL;
[0018] (2) Add the Myoglobin solution system in the above step (1) to a 30K ultrafiltration tube, and centrifuge at 10000g for 30min;
[0019] (3) Add 100 μL of 50 mM Tris-HCl, 150 mM NaCl, 20 mM CaCl to the ultrafiltration tube of the above step (2) 2 solution, the pH is 5.0, the Urea concentration is lower than 2M, the ratio of protein to enzyme is 20:1, add 10 μg of the protease, and incubate overnight at 20°C;
[0020] (4) Centrifuge the ultrafiltration tube with the Myoglobin solution after enzymolysis in the above step (3) at 10000 g for 30 min, and collect about 100 μL of proteolysis solution;
[0021] (5) Add 100 μL of 50 mM Tris-HCl, 150 mM NaCl, 20 mM CaCl to the ult...
Embodiment 2
[0025] Hydrolysis of bovine serum albumin (BSA) with short-chain hydrophobic amino acid specific protease and detection
[0026] (1) Take 0.2mg of BSA, dissolve in 200μL of 200mM Tris-HCl, 8M Urea, 200mMNaCl, 1mMCaCl 2 In the solution, the pH is 9.0, so that the BSA concentration in the solution is 1mg / mL;
[0027] (2) Add 500mM DTT 16μL to the BSA solution obtained in the above step (1) to make the final concentration of DTT 40mM, incubate at 100°C for 5min, then add 500mM IAA32μL, the final concentration is 80mM, and incubate at 25°C in the dark for 40min , to open the disulfide bond;
[0028] (3) Add the BSA solution system processed in the above step (2) into a 30K ultrafiltration tube, and centrifuge at 10000g for 30min;
[0029] (4) Add 100 μL of 200 mM Tris-HCl, 200 mM NaCl, 1 mM CaCl to the ultrafiltration tube of the above step (3) 2 solution, the pH is 9.0, the concentration of Urea is lower than 0.5M, the ratio of protein to enzyme is 50:1, add 4 μg of the protea...
Embodiment 3
[0035] The proteolytic enzyme Scanase digests the whole protein of HeLa cells and detects:
[0036] (1) HeLa cells were lysed to extract 0.2 mg of whole protein, with a volume of 200 μL, added to a 30K ultrafiltration tube, and centrifuged at 10,000 g for 30 min;
[0037] (2) Add 200μL of 50mM Tris-HCl, 8M Urea, 150mMNaCl, 20mM CaCl to the ultrafiltration tube of the above step (1) 2 Solution, pH 8.0, centrifuged at 10000g for 30min;
[0038] (3) repeat above-mentioned step (2) twice again;
[0039] (4) Add 200 μL of 50 mM Tris-HCl, 150 mM NaCl, 20 mM CaCl to the ultrafiltration tube of the above step (3) 2 Solution, pH 8.0, centrifuged at 10000g for 30min;
[0040] (5) repeat above-mentioned step (4) twice again;
[0041] (6) Add 100 μL of 50 mM Tris-HCl, 150 mM NaCl, 20 mM CaCl to the ultrafiltration tube of the above step (5) 2 solution, pH 8.0;
[0042] (7) Add 8 μL of 500 mM DTT to the whole protein solution of HeLa cells obtained in the above step (6) to make the f...
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