Methods of treating neurological disorders
A neurological and disease-related technology, applied in the field of neurological disease treatment, can solve problems such as the destruction of tau's normal structure and function
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Embodiment 1
[0162] Example 1. Titration of protease activity
[0163] This example shows the titration of the protease activity of elastase, cathepsin G and trypsin using casein as the reaction substrate.
[0164] Prepare 1.0 mg / ml in Phosphate Buffered Saline (PBS) Labeled casein stock solution, then in digestion buffer (10mM Tris-HCl, pH 7.8, 0.1mM NaN 3 ) diluted to 10 μg / ml. Dilute elastase, cathepsin G, or trypsin to various concentrations in digestion buffer, and add 100 μl of the diluted protease to each well of a 96-well microplate. 100 μl of 10 μg / ml Labeled casein was added to each sample well and incubated in the dark for 1 hour. After incubation, read the microplate using a fluorescent microplate reader.
[0165] like figure 1 As shown, the elastase, cathepsin G, and trypsin enzyme preparations all exhibited casein digestion activity with linear reaction kinetics up to about 6 μg / ml of each protease.
Embodiment 2
[0166] Example 2. Digestive activity of protease immobilized on an agarose resin column
[0167] This example shows the protease activity of elastase, cathepsin G and trypsin immobilized on NHS-activated agarose resin using casein as the reaction substrate.
[0168] NHS-activated agarose resin columns were prepared according to the manufacturer's instructions. Columns consisted of 100 μl of prewashed agarose resin with 200 μl of control buffer, or 2 mg / ml of elastase, cathepsin G, trypsin, kallikrein 5 (KLK5), or lysyl endopeptidase-activated kallikrein Enzyme 6 (KLK6). Spin the column and collect the flow-through to determine the efficiency of protease immobilization. No protein was detected in the flow-through as determined by Bradford (data not shown). Low protease activity (determined by the protease activity assay described in Example 1) was detected in the flow-through, indicating a coupling efficiency of >99.9%. Block remaining free NGS sites by adding 1 M ethanolam...
Embodiment 3
[0171] Example 3. Digestion of Tau, α-synuclein and TDP-43 by proteases immobilized on an agarose resin column
[0172] This example shows the protease activity of resin-immobilized elastase, cathepsin G and trypsin using Tau and α-synuclein as reaction substrates. The Tau protein forms tested included the 2N4R isoform with a P301L substitution (“Tau-441(P301L)”), the 2N3R isoform (“Tau-410”), and the 2N4R isoform phosphorylated by GSK3β (“p- Tau-441").
[0173] To assess protease activity against Tau-441(P301L), 100 μl of a 50 μg / ml Tau(P301L) solution was added to a blank column or a column immobilized with elastase, cathepsin G, or trypsin (as described in Example 2 preparation). The columns were incubated on a rotating stand at room temperature or in a thermostatic mixer at 37°C. At incubation times of 2, 10 and 20 minutes, the columns were centrifuged for 10 seconds and 20 μl samples were removed for SDS-PAGE analysis. 16 μl / 0.8 μg of protein was loaded onto a 10% pol...
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