A screening method for dihydrolipoic acid succinyltransferase inhibitors
A technology of dihydrolipoic acid succinyl and screening method, which is applied in the field of biochemistry and can solve the problems of lack, difficulty in research and development of DLST inhibitors, etc.
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Embodiment 1
[0080] Example 1: Methanesulfonyl ipconazole rice bacterial leaf blight (of Xoo) of total protein in vitro screening results
[0081] Take 100mL OD 600 Xoo bacilli = 0.6, centrifuged at 7500rpm 10min, the supernatant discarded, and then washed three times with PBS. Take 10mL PBS (pH = 7.2) the cells were resuspended, centrifuged tubes on ice sonicated, then diluted to 1mg / mL as measured by protein concentration bradford. Take 43μL of total protein, was added to different concentrations 1μL methanesulfonyl ipconazole, the final concentration of 50,100,250,500μM, an equal volume of DMSO control group, were incubated for 2h at 25 ℃. 5μM probe 1 then were added, and incubation continued for 2h. Biotin-N were added to the system 3 , Sodium ascorbate and BTTAA / CuSO 4 Click reaction mixed solution. 2h then 2 × loading buffer were added to the system, boiled for 10min at 95 ℃. 10μg samples were taken for SDS-PAGE, and then transferred to a membrane at 200mA constant current 50min, PBS...
Embodiment 2
[0082] Example 2: Methanesulfonyl ipconazole citrus canker (of Xac) of total protein in vitro screening results
[0083] Take 100mL OD 600 Bacteria Xac = 0.6, centrifuged at 7500rpm 10min, the supernatant discarded, and then washed three times with PBS. Take 10mL PBS (pH = 7.2) the cells were resuspended, centrifuged tubes on ice sonicated, then diluted to 1mg / mL as measured by protein concentration bradford. Take 43μL of total protein, was added to different concentrations 1μL methanesulfonyl ipconazole, the final concentration of 10,25,50,100,250μM, an equal volume of DMSO control group, were incubated for 2h at 25 ℃. Then 50μM probe 2 were added, and incubation continued for 2h. Biotin-N were added to the system 3 , Sodium ascorbate and BTTAA / CuSO 4 Click chemistry reaction mixture solution. 2h then 2 × loading buffer were added to the system, boiled for 10min at 95 ℃. 10μg samples were taken for SDS-PAGE, and then transferred to a membrane under 50min at 200mA, PBST 2h blo...
Embodiment 3
[0084] Example 3: Methanesulfonyl ipconazole vitro screening results for recombinant expression DLST
[0085] After expanding the culture of pet28a vector to construct a recombinant plasmid which was introduced into the BL21 strain, the Ni-column was then affinity purified by thrombin digestion, recombinant DLST finally purified by molecular sieve. 25ng taken recombinant DLST, was added biotin-N 3 , Sodium ascorbate and BTTAA / CuSO 4 Click chemistry reaction mixture solution. After an additional 2 × loading buffer were added to the system boiled 10min at 95 ℃. 12.5ng recombinant DLST were taken for SDS-PAGE, and then transferred to a membrane under 50min at 200mA, PBST 2h blocked with 5% skim milk after washing the membrane. The membranes were incubated 2h with HRP-labeled rear of PBST chain streptavidin (HRP-Streptavidin), followed by addition of HRP-labeled streptavidin streptavidin (HRP-Streptavidin) were incubated for 2h, chemiluminescent after PBST the membrane was washed Fi...
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