Preparation method of urinary protein and detection method for urinary proteome
A urine protein and protein technology, which is applied in the detection field of urine proteome, can solve the problems of low throughput of quantitative deep urine proteome, etc., and achieve the effects of improving accuracy and repeatability, short mass spectrometry detection time, and simple preparation process
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[0055] Example 1 Preparation method of urine protein
[0056] (1) 1ml urine sample was centrifuged at 20°C for 75 minutes with a centrifugal force of 200,000 g, the supernatant was discarded, and the precipitate was retained;
[0057] (2) Transfer the precipitate to a 1.5 ml centrifuge tube, add 400 μl of resuspension buffer (50 mM Tris, 250 mM sucrose, pH 8.5) to the centrifuge tube, let it stand at room temperature for 30 minutes, and pipette fully Suspended sediment
[0058] (3) Add dithiothreitol to the above-mentioned resuspended precipitate to a final concentration of about 100mM, and heat at 65°C for 30 minutes to remove most of the uromodulin (UMOD, see figure 1 );
[0059] (4) Add washing buffer (10mM triethanolamine, 100mM sodium chloride, pH7.4) to 4ml, then centrifuge at 200,000g at 20°C for 75 minutes, discard the supernatant, and leave the precipitate;
[0060] (5) Re-dissolve the precipitate with 30 μl digestion buffer (such as 10 mM Tris or ammonium bicarbonate, with or...
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[0064] Example 2 Preparation method of urine protein
[0065] (1) A 10ml urine sample was centrifuged at a centrifugal force of 100,000 for 20 minutes at 4°C, the supernatant was discarded, and the precipitate was retained;
[0066] (2) Transfer the above-mentioned precipitate to a centrifuge tube, add 60ul of resuspension buffer (50mM Tris, 250mM sucrose, pH8.5) to the centrifuge tube, let stand at room temperature for 10 minutes, and pipette to resuspend the precipitate. ;
[0067] (3) Add dithiothreitol to the above resuspended precipitate to a final concentration of 50mM, and heat at 80°C for 10 minutes to remove most of the uromodulin in the sample;
[0068] (4) Add cleaning buffer (10mM triethanolamine, 100mM sodium chloride, pH7.4) to 400ul, then centrifuge for 20 minutes under 4 conditions at a centrifugal force of 100,000, discard the supernatant, and save the precipitate;
[0069] (5) Use one-dimensional electrophoresis (SDS-PAGE) for protein separation, dissolve the precipit...
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[0070] Example 3 A method for enriching urine protein,
[0071] Including the following steps:
[0072] (1) Add 10 mg of diatomaceous earth to 1 ml of urine and shake well;
[0073] (2) Heat at a temperature between 37-100°C for 5 minutes, and cool to room temperature;
[0074] (3) Rotate and mix at room temperature for 30 minutes, centrifuge at 12000 rpm for 5 minutes, and discard the supernatant;
[0075] (4) Add 30-100 microliters of digestion buffer, and carry out digestion in the solution enriched in urine protein.
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