Real Thiol

• CAT Number: I046319
• CAS Number: 2280796-90-7
• Molecular Formula: C20H17N3O7
• Molecular Weight: 411.36
• Purity: ≥95%
• Synonyms: 2-[[(E)-3-[7-(azetidin-1-yl)-2-oxochromen-3-yl]-2-cyanoprop-2-enoyl]-(carboxymethyl)amino]acetic acid
• InChI: InChI=1S/C20H17N3O7/c21-9-14(19(28)23(10-17(24)25)11-18(26)27)7-13-6-12-2-3-15(22-4-1-5-22)8-16(12)30-20(13)29/h2-3,6-8H,1,4-5,10-11H2,(H,24,25)(H,26,27)/b14-7+
• InChIKey: VNKDTNMOOIIYSL-VGOFMYFVSA-N
• SMILES: C1CN(C1)C2=CC3=C(C=C2)C=C(C(=O)O3)C=C(C#N)C(=O)N(CC(=O)O)CC(=O)O

Real Thiol is a reversible reaction-based fluorescent probe which can quantitatively monitor the real-time glutathione dynamics in living cells.
Real Thiol (RT) shows ratiometric fluorescence responses with a wide dynamic range when reacting with Glutathione (GSH). Real Thiol and its GSH adduct (RT-GSH) shows fluorescence maxima at 487 and 562?nm with excitation wavelengths at 405 and 488?nm, respectively. The Kd for the reaction between Real Thiol and GSH is 3.7?mM. Real Thiol has much improved quantum yields and photostability. The wide dynamic range of Real Thiol allows for monitoring of the GSH level changes within 1 to 10?mM in both directions in living cells. Gel permeation chromatography (GPC) analysis showes that 10% of Real Thiol reacts with thiolated proteins while 90% of Real Thiol reacts with GSH[1].
Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs)[1].
1. Real Thiol (RT) is dissolved in DMSO to obtain a 10 mM stock solutions and further diluted before use.
2. Cells are cultured in 10% FBS DMEM containing different concentrations of Buthionine sulfoximine (0.9, 1.9, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250 and 500?μM) for 72?h and harvested for analysis.
3. Cells are then suspended in fresh medium containing 1?μM of Real Thiol probe as single-cell suspensions, and keep the staining time between 9 and 10?min for each sample.
4. Analyze sample on a flow cytometer. PE and pacific-blue channels were selected to measure the fluorescence intensities of unreacted and reacted Real Thiol, respectively.

Reference

[1]. Jiang X, et al. Quantitative real-time imaging of glutathione. Nat Commun. 2017 Jul 13;8:16087.
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