Pharmaceutical application of hydrogen sulfide synthase CSE
A technology for synthesizing enzymes and hydrogen sulfide, applied in the field of biomedicine, to achieve the effect of improving endothelial injury, reducing aortic aneurysm and aortic dissection
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[0018] 1. Western-Blot
[0019] (1) SDS-PAGE (polyacrylamide gel) electrophoresis: Prepare 12% separating gel and 3% stacking gel. Take 15 μL sample solution and 3 μL 6× loading buffer, and mix well. After boiling for 5 min to denature the protein, the sample was loaded with an amount of about 30 μg per well. Electrophoresis at a constant voltage of 110 V for about 90 min until bromophenol blue completely disappeared.
[0020] Preparation of SDS-PAGE:
[0021] Components Separating Gel (12%) Stacking Gel (3%)
[0022] Deionized water 1.6 mL 1.8 mL
[0023] 30% AA stock solution 2.0 mL 0.3 mL
[0024] 1.5M Tris-HCl (pH8.8) 1.3 mL ---
[0025] 1 M Tris-HCl (pH6.8) --- 0.75 mL
[0026] 10% SDS 50 μL 30 μL
[0027] 10% ammonium persulfate (APS) 50 μL 50 μL
[0028] TEMED 5 μL 5 μL
[0029] Total volume 5 mL 3 mL
[0030] (2) Membrane transfer: After electrophoresis, cut off the stacking gel, soak the gel in protein transfer buffer (3.6 g / L Tris, 300 mL / L methanol, 17.3 ...
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