A method for large-scale screening of protein biomarkers
A biomarker and protein technology, applied in the field of molecular biology, can solve the problems of small dynamic range, poor consistency, and high cost, and achieve the effects of low cost, large number of screenings, and accurate screening results
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Embodiment 1
[0026] 4 g of mouse liver tissue control samples and 4 g of pathological samples were cut into pieces and transferred to a homogenizer. PMSF / isopropanol stock solution (2 mL) was added to the pre-chilled lysis buffer. Quickly add 2 mL of pre-chilled lysis buffer to the homogenizer, and grind thoroughly in an ice bath. The tissue grinding fluid was transferred to a 1.5 mL centrifuge tube and centrifuged at 4°C (14000 rpm, 10 min). After centrifugation, suck the supernatant and transfer it to a new 1.5mL centrifuge tube, which is the crude tissue protein extract. The obtained protein extract can be stored at -20°C for subsequent experiments.
[0027] The above-mentioned protein extracts were spotted with a spotter and fixed in 96-well plates to react for 1 hour. The plate was blocked with a solution containing skim milk to prevent non-substantial substances from sticking to the chip. Take out the material in the orifice plate, crush the glue with a glass rod, dissolve it wit...
Embodiment 2
[0054] Take 4g of mouse spleen tissue control sample and 4g of pathological tissue and put it in 4ml of protein lysis solution A (10mmol / LMgCl2, 1mmol / LKCl, 1mmol / L phenyltetraylsulfonyl fluoride, 1mmol / L dithiothreitol), homogeneously Slurry until the particles are invisible to the naked eye, add a few drops of 3 mmol / L sucrose to mix well, centrifuge at 10,000 r / min at 4°C for 30 min, and discard the supernatant. Dissolve the precipitate in 3-5 times protein lysis solution (0.5mol / LKCl) and mix well. After centrifugation at 10000r / min for 30 min at 4°C, take the supernatant and store it at -80°C for later use.
[0055] The above-mentioned protein extracts were spotted with a spotter and fixed in 96-well plates to react for 1 hour. The plate was blocked with a solution containing skim milk to prevent non-substantial substances from sticking to the chip. Take out the material in the orifice plate, crush the glue with a glass rod, dissolve it with buffer solution (0.3% bromoph...
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