Cuso4

Cells were pretreated with 20 μM photo-affinity probe 496 or photostable control compound 484 for 1 h, and then lysates were prepared at a concentration of 2 mg/mL total protein in Nonidet P-40 buffer. The whole-cell lysates, or the purified hRIPK1 (residues 1–330) proteins were individually treated with 200 μM 484 or 496 for 30 min, and all samples were photo-crosslinked at ∼350 nm on ice for 30 min using a UV crosslinker (energy: 1200). After photo-affinity labeling, click chemistry was preformed to allow the linkage of Biotin-PEG3-Azide to enable pulldown^{26 (link)}. A master mix of the catalyst (final concentration) was prepared immediately before use by combining: 100 μM Biotin-PEG3-Azide (TCI (Shanghai) Development Co.,Ltd.), 100 μM TBTA (TCI (Shanghai) Development Co.,Ltd.), 1 mM CuSO₄ (Sinopharm Chemical Reagent Co.,Ltd.) 1 mM CuBr (Shanghai Macklin Biochemical Technology Co.,Ltd.), 1 mM TCEP (Sun Chemical Technology (Shanghai) Co.,Ltd.). The samples were vortexed and incubated for 1 h at room temperature and TCEP was added one more time after incubation for 30 min. Chloroform-methanol precipitation was preformed next to isolate proteins. The biotin-labeled proteins were isolated by incubating with streptavidin-coupled beads overnight at 4 °C.

ASC, GA, Tyr, acid chrome blue K (ACBK), sulfanilic acid (ABSA) and glutamate (GA) were purchased from Aladdin Chemical Co. Ltd., (Shanghai, China). HAuCl₄ and CuSO₄ were obtained from Sinopharm Chemical Reagent Co. Ltd., China. Phosphate buffer solution (PBS, 0.1 M) was prepared by mixing stock solutions of 0.2 M Na₂HPO₄·12H₂O and NaH₂PO₄·2H₂O. All reagents were of analytical grade and were used directly without purification, and all solutions were prepared with double-distilled water.
A pH meter (FE28-FiveEasy Plus, METTLER TOLEDO), electronic analytical balance (BS224S, Sartorius), ultrasonic cleaner (SK1200H, KUDOS), scanning electron microscope (SU8010, HITACHI, Japan) and magnetic stirrer (DF-101Z) were used. The fabrication of modified electrodes and rice wine identified experiments were performed on a PARSTAT 3000 A electrochemical workstation (AMETEK. Inc, USA). A conventional three-electrode system consisted of a working modified electrode, a platinum wire auxiliary electrode and a saturated Ag/AgCl reference electrode. A saturated KCl solution was used as the reference solution.

Chlorauric acid (HAuCl₄∙3H₂O), potassium tetrachloroplatinate (II) (K₂PtCl₄), cetyltrimethylammonium bromide (CTAB), hydrogen peroxide, L-ascorbic acid (AA), 3,3′,5,5′-tetramethylbenzidine (TMB), cysteine, L-glutathione reduced (GSH), L-glutathione oxidized (GSSG), horseradish peroxidase and sodium hydrosulfide (NaHS) were purchased from Alfa Aesar. FeSO₄∙6H₂O, Co(NO₃)₂∙6H₂O, ZnSO₄, CuSO₄, Mg₂SO₄, Na₂S∙9H₂O, glucose, uric acid and glycine were obtained from Sinopharm Chemical Reagent Co., Ltd. Milli-Q water (18 MΩ cm) was used in the preparation of all solutions.

Grape seeds were purchased from Chinese pharmacy (Yangzhou, China). Quinine sulfate was purchased from Aladdin (Shanghai, China). CuSO₄, FeCl₃·6H₂O, FeSO₄, CoCl₂, AgNO₃, Pb(NO₃)₂, ZnSO₄, Cr(NO₃)₃, NiSO₄, MgCl₂, CaCl₂, MnCl₂, AlCl₃, NaCl and CH₄N₂S were purchased from Sinopharm Chemical Reagent Co, Ltd (Shanghai, China). All reagents were of analytical reagent grade.

PVA, H₂SO₄, H₂O₂, HCl and CuSO₄ were purchased from Sinopharm Chemical Reagent, China. GO was prepared from natural graphite powder using the modified Hummers method. Deionized water with a resistivity of 18.1 MΩ cm was used for all experiments.