NBD-Cl is a nonfluorescent reagent which becomes highly fluorescent after reaction with thiol or amino groups^[1].

NBD-Cl (NBD chloride) forms highly fluorescent derivatives for detection of all the protein amino acids. In addition, NBD-Cl provides a simple and sensitive method for determination of N-terminal amino acids. The differences in intensity and color of fluorescence can be used to advantage to identify prolyl peptides^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

199.55

C₆H₂ClN₃O₃

10199-89-0

Room temperature in continental US; may vary elsewhere.

4deg;C, protect from light

*In solvent : -80deg;C, 6 months; -20deg;C, 1 month (protect from light)

DMSO : ≥ 100 mg/mL (501.13 mM)

H₂O : 0.67 mg/mL (3.36 mM; Need ultrasonic)

* “≥” means soluble, but saturation unknown.

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month (protect from light)。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂：;10% DMSO ;; 90% (20% SBE-β-CD in saline)

  Solubility: ≥ 2.5 mg/mL (12.53 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (12.53 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明

• [1]. Fager RS, et al. The use of NBD chloride (7 chloro-4-nitrobenzo-2-oxa-1,3-diazole) in detecting amino acids and as an N-terminal reagent. Anal Biochem. 1973 May;53(1):290-4.

Fluorescence Measurements^[1]
Fluorescence spectra are recorded on a Hitachi MPF-24 Fluorescence Spectrophotometer. The emission spectrum is determined at the optimal wavelength of 464 nm. Thin-layer chromatography is carried out on Brinkmann glass supported analytical silica G thin-layer plates without fluorescent indicator. The NBD derivatives are visualized on thin-layer plates either by examination under an ultraviolet lamp with a long wavelength filter or by using the same lamp with a 2×2-in. Corning 5-57 filter and viewing the spots through a 2×2-in. Corning 3-68 filter. The former method produces a more intense fluorescence, but the latter produces a much greater contrast appearing as white spots against a dark background. NBD-Cl (NBD chloride) is allowed to react with amino acids and peptides under the conditions. Into 3 mL Pyrex test tubes are pipette the following: (1) 2.0 mL of ethanol, (2) 0.1 mL NBD-Cl solution in ethanol 1.41 pg/mL, (3) 0.1 mL ethanol saturated with sodium acetate, (4) 0.1 mL of amino acid solution 400-600μg/mL, and the mixture incubated at 75°C for 20 min. For reaction with peptides, 1.5 mL of ethanol, 0.1 mL ethanolic sodium acetate, 0.4 mL ethanolic NBD-Cl, 1.41 μg/mL, are added to 0.4 ml of the peptide and incubated under the same conditions.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Fager RS, et al. The use of NBD chloride (7 chloro-4-nitrobenzo-2-oxa-1,3-diazole) in detecting amino acids and as an N-terminal reagent. Anal Biochem. 1973 May;53(1):290-4.