Application of fiber gelatinized protein 3
A technology of colloidal gold test paper and diabetes, which is applied in the application field of protein molecular markers, and can solve the problems such as no reports on the correlation of diabetic nephropathy
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Embodiment 1
[0021] Example 1 , normal healthy controls and the preparation of serum samples from patients with type 2 diabetes mellitus
[0022] Sodium phosphate used in this example was purchased from Sigma Company, formic acid was purchased from Fluka Company, and acetonitrile was purchased from Merck Company.
[0023] Serum samples from normal healthy controls and patients with type 2 diabetes were provided by the Diabetes Institute of Shanghai Sixth People's Hospital, and the protein concentration in the original plasma was quantified by the Bradford method to be about 100 mg / mL.
[0024] The original serum was diluted to 20 mg / mL with 50 mM sodium phosphate buffer solution (pH 2.5, containing 5% acetonitrile), and passed through a 0.22 μm filter membrane at room temperature at a speed of 10,000 g / min. The resulting solution was replaced by dialysis into solution A of strong cation exchange chromatography, namely 50 mM sodium phosphate buffer solution (pH 2.5, containing 5% acetonit...
Embodiment 2
[0030] Example 2 1. Screening method for differentially expressed proteins
[0031] Dithiothreitol (DTT) used in this example was purchased from Sigma; iodoacetamide (IAA) and formic acid were purchased from Fluka; acetonitrile was purchased from Merck; capillary reversed-phase liquid chromatography was purchased from Agilent.
[0032] The composition of the loading buffer used was: 0.6 mL of 1 mol / L Tris-HCl (pH6.8), 5 mL of 50% glycerol, 2 mL of 10% SDS, 0.5 mL of mercaptoethanol, 1.9 mL of distilled water, and a small amount of bromophenol blue.
[0033] Take equal amounts of 1.8 mg of mixed serum samples from diabetic patients and normal healthy controls (from 5 individuals each), add 1 μL 1M DTT to them, and place them at 56°C for 30 min. After cooling to room temperature, add 5 μL 1M IAA, Store at room temperature in the dark for 40 minutes.
[0034] The above-mentioned treated plasma samples were separated by one-dimensional gel electrophoresis (7.5-17.5% gradient ge...
Embodiment 3
[0058] Example 3 , Screening method for differentially expressed proteins 2
[0059] Dithiothreitol (DTT) used in this example was purchased from Sigma; iodoacetamide (IAA) and formic acid were purchased from Fluka; acetonitrile was purchased from Merck; capillary reversed-phase liquid chromatography was purchased from Agilent.
[0060] Add 250 μL solution (100 mM NaCl, 10 mM HEPES, pH 7.4) to 50 μL serum sample, use a 0.22 μm filter membrane to remove lipid by centrifugation at 10 000 g at 4°C; take 260 μL lipid-free serum sample and add 182 μL cold ethanol solution , placed on a vertical shaker at 4°C for 1 h, and then centrifuged at 16 000 g for 45 min at 4°C. The supernatant is the high-abundance component, and the precipitated part is the low-abundance component.
[0061] Both components were lyophilized, and then 200 μL of 2D lysate was added for dissolution. Take 100 μL of high-abundance and low-abundance fractions, add 1 μL 1M DTT, mix well, place at 37°C for 2.5 h...
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