Protease substrate screening method based on solid-loaded mixed protein as screening database
A screening method, protease technology, applied in biochemical equipment and methods, immobilized on/in organic carriers, determination/inspection of microorganisms, etc. Simple, specific and reliable results
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[0033] Example 1 Screening of caspase 3 substrates based on agarose microspheres as a carrier.
[0034] Preparation of protein samples: Proteins from human acute T-cell leukemia cells (Jurkat) were extracted by non-denaturing lysis. 10 8 Add 4 mL of ice-bathed non-denaturing lysate to each Jurkat cell, and the composition of the lysate is as follows: 50 mM 4-hydroxyethylpiperazineethanesulfonic acid (pH=7.4), 1 mM dithiothreitol, 150 mM sodium chloride, 1% ( v / v) Nonionic surfactant Triton X-100, 1mM ethylenediaminetetraacetic acid, 2% (v / v) protease inhibitor cocktail (Sigma), 2 / 5 tablets of phosphatase inhibitor (PhosSTOP , Roche), 1 mM phenylmethylsulfonyl fluoride. The ultrasonic power in the ice bath was 400W, the ultrasound was performed for 3s, the interval was 5s, and the ultrasound was repeated 180 times. Then centrifuge at 16000g centrifuge for 20min at 4°C, take the supernatant protein, measure the protein concentration to 6.5mg / mL according to the BCA protein co...
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