Naph-EA-mal (Thiol-green 1) is a rapid detection and ultrafast turn-on thiol (thiol) fluorescent probe that can be used for protein labeling and bioimaging. Naph-EA-mal (Thiol-green 1) may be utilized to detect thiols in living cells, label protein thiols, quantify total thiol concentrations in cell lysates, and determine reversible protein thiol oxidation in fixed cells. Ex: 488 nM; Em: 540 nM.

Physicochemical Properties

Molecular Formula	C22H21N3O4
Molecular Weight	391.42
Exact Mass	391.153
CAS #	210292-65-2
PubChem CID	132564217
Appearance	Light yellow to orange solid powder
LogP	2.6
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	7
Heavy Atom Count	29
Complexity	716
Defined Atom Stereocenter Count	0
SMILES	N(C1=CC=C2C(N(CCCC)C(=O)C3C=CC=C1C2=3)=O)CCN1C(C=CC1=O)=O
InChi Key	RWRDYDLQZILWMW-UHFFFAOYSA-N
InChi Code	InChI=1S/C22H21N3O4/c1-2-3-12-25-21(28)15-6-4-5-14-17(8-7-16(20(14)15)22(25)29)23-11-13-24-18(26)9-10-19(24)27/h4-10,23H,2-3,11-13H2,1H3
Chemical Name	2-butyl-6-[2-(2,5-dioxopyrrol-1-yl)ethylamino]benzo[de]isoquinoline-1,3-dione
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro

Using Naph-EA-mal (Thiol-green 1) (1 μM; 5 mins) on Hep G2 cells, it is possible to image thiols in living cells [1]. A strong green fluorescence is seen. Less fluorescence is seen in cells when they are preincubated with NEM (a thiol block reagent; 100 μM) to inhibit the intracellular thiols[1]. Monitoring thiol-disulfide levels in fixed cells Naph-EA-mal (Thiol-green 1) (10 μM) causes high fluorescence in Hep G2 cells because most cellular thiols are present in the reduced form. Sharp dimming of the fluorescence signal is seen following diamide treatment[1]. After diamide (200 μM) oxidized the Hep G2 cells for 30 minutes, 70% EtOH fixed them, and NEM (100 μM) inhibited the residual free thiols. Subsequently, the cells undergo further treatment with TCEP (5 mM), and Naph-EA-mal (Thiol-green 1) (10 μM) is added. Once more, the green fluorescent signal is observed[1].

References

[1]. An ultrafast turn-on thiol probe for protein labeling and bioimaging. Analyst.

Solubility Data

Solubility (In Vitro)

DMSO: 33.33 mg/mL (85.15 mM)

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.5548 mL	12.7740 mL	25.5480 mL
	5 mM	0.5110 mL	2.5548 mL	5.1096 mL
	10 mM	0.2555 mL	1.2774 mL	2.5548 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.