Final concentrations of 1 mM DAz-2 (Cayman Chemical), 1 mM Cyanine5 alkyne (Lumiprobe), 2 mM copper(II)-TBTA complex (Lumiprobe) and 4 mM ascorbic acid made in situ, were added sequentially in 15 mM PBS buffer mixed with 30% (vol/vol) acetonitrile. The solution was mixed at RT overnight and then quenched with 20 mM ethylenediaminetetraacetic acid (EDTA) and mixed at RT for 2 hr.
Daz 2
Manufactured by Cayman Chemical
Sourced in United States
DAz-2 is a laboratory equipment product manufactured by Cayman Chemical. It is designed for use in scientific research and analytical applications. The core function of DAz-2 is to provide a reliable and precise method for the detection and quantification of specific analytes in a sample.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using daz 2
1
Copper-catalyzed Azide-Alkyne Cycloaddition
2
Labeling Reversible Cysteine Oxidation
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The method used to label RSOH in ThrRS was modified from previous publications (36 (link),37 (link)). Briefly, purified WT and mutant ThrRSs were first treated with 10 mM dithiothreitol (DTT) at room temperature for 20 min to reduce any pre-existing reversibly oxidized forms of thiol group, including disulfide bonds and RSOH. After removing DTT with Bio-Spin 6 desalting columns (Bio-Rad), 5 μM or 2 μM protein was incubated with oxidants at different molar ratios and 40-fold excess of DAz-2 (Cayman Chemical) at 37°C for 1 h. In the experiments testing the effects of metals on RSOH formation, protein samples were incubated with 5-mM ethylenediaminetetraacetic acid (EDTA) on ice for 30 min. EDTA was then removed with Bio-Spin 6 columns before DAz-2 labeling in the presence and absence of metals.
3
Sulfenic Acid Detection in Oxidized cpEF-Tu
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Sulfenic acid in oxidized cpEF-Tu was allowed to react with DAz-2 (Cayman Chemical Co., Ann Arbor, MI, U.S.A.), a reagent that is specific for sulfenic acid, and the product was visualized by immunoblotting analysis with streptavidin-conjugated horseradish peroxidase, as described previously [7 (link)].
4
Biotin Switch Assay for Protein S-Nitrosation
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A mixture of sodium hydrogen sulfite and sodium sulfite (NaHSO3 [243973-100G, Sigma, USA]/Na2SO3 [S4672-250G, Sigma, USA], freshly mixed in a 1:3 ratio, pH 7.4), was used as the SO2 donor. Tamoxifen (579002, Sigma, USA) in corn oil was used to induce the recombination activity of Cre. The main reagents for cell culture included the following: FBS (FB25015, CLARK Bioscience, USA), 100 × PS (15140-122, Thermo Scientific, USA), 100×glutathione (LG) (25030081. Thermo Scientific, USA), and trypsin containing EDTA (0.25%) (25200-056, Thermo Scientific, USA). DAz-2 (13382, Cayman, USA), p-biotin (13581, Cayman, USA), and hyperlinked Neutral Affinity ProteinTM (53150, Thermo Fisher Science, USA) were used for biotin switch assay. The antibodies used in this study was listed in Supplementary Table 1 .
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