Coumarin dyes, coumarin 525 and coumarin 545, were purchased from Exciton (Dayton, OH, USA). Amino acids—DL-proline (Pro), DL-valine (Val), DL-isoleucine (Ile), DL-leucine (Leu), and DL-phenylalanine (Phe)—were obtained from Sigma-Aldrich (St. Louis, MO, USA). The fluorescence derivatization reagent NBD-F was purchased from Dojindo Molecular Technologies (Kumamoto, Japan), and trifluoroacetic acid (TFA) was sourced from Wako Pure Chemicals (Osaka, Japan). Dimethyloctadecylchlorosilane was purchased from Tokyo Chemical Industry (Tokyo, Japan). Acetonitrile (HPLC-grade) was obtained from Merck KGaA (Darmstadt, Germany). A Milli-Q system (Merck KGaA) was used for water purification.
Nbd f
Manufactured by Dojindo Laboratories
Sourced in Japan
NBD-F is a fluorescent labeling reagent used in analytical chemistry. It is primarily employed in the derivatization of primary and secondary amines for subsequent detection and quantification using fluorescence spectroscopy.
Automatically generated - may contain errors
Lab products found in correlation
Hplc grade acetonitrile, by Merck Group (2 mentions)
Milli q system, by Merck Group (2 mentions)
Sbd f, by Dojindo Laboratories (2 mentions)
Trifluoroacetic acid tfa, by Fujifilm (1 mentions)
Synapt g2 s mass spectrometer, by Waters Corporation (1 mentions)
Acquity uplc system, by Waters Corporation (1 mentions)
Chloroform, by Avanti Polar Lipids (1 mentions)
Methylamine, by Fujifilm (1 mentions)
Amino acid, by Merck Group (1 mentions)
6 protocols using nbd f
1
Fluorescence Labeling of Amino Acids
2
HPLC Separation of Nectrisine and 4-Amino-4-deoxyarabinitol
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To detect and efficiently separate nectrisine and 4-amino-4-deoxyarabinitol by HPLC, samples were labeled using 4-fluoro-7-nitrobenzofurazan (NBD-F) (Dojindo Laboratories, Kumamoto, Japan) (Watanabe and Imai 1981 (link); Imai and Watanabe 1981 (link)). Cell extracts (20 μL) were reduced with 10 μL of NaBH4 solution (1 mg/mL) to convert nectrisine to 1,4-Dideoxy-1,4-imino-d -arabinitol (DAB). The resulting solutions were heated at 60 °C for 2 min with 60 μL of 2 g/L NBD-F in methanol and 10 μL of 100 mM borate buffer, pH 8.2. The solutions were cooled to room temperature and acidified with 10 μL of 0.5 M HCl aqueous solution. LC–MS analysis was conducted using an Acquity UPLC System and a Synapt G2-S Mass Spectrometer (Waters). NBD-labeled samples were injected into a Unison UK C18 4.6 × 150 mm column (Imtakt, Kyoto, Japan) at a flow rate of 1 mL/min at 30 °C. The mobile phase consisted of solvent A, 10 mM NH4CO2H/2.2 mM HCO2H in H2O; and solvent B, 2.2 mM HCO2H in CH3CN, using the gradient elution of, 10 % B for 8 min, from 10 to 90 % B for 20 min, and 90 % B for 2 min.
3
Quantification of Amino Acids in Mice Serum and CSF
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Male mice (8–11 weeks of age) were anesthetized with isoflurane inhalation. Blood was collected by cardiocentesis, and serum was prepared after coagulation and centrifugation. CSF samples were collected by ventricular puncture with glass capillaries. Serum and CSF amino acid levels were measured using high-performance liquid chromatography (HPLC), amino acid labeling with 4-fluoro-7-nitrobenzofurazn (NBD-F; Dojindo, Kumamoto, Japan), and thiol labeling with 4-fluoro-7-sulfobenzofurazn (SBD-F; Dojindo) as previously described [28 (link),29 (link)]. NBD-F labeling of tryptophan (Trp) resulted in a fluorescence wave change, and Trp was detected by its fluorescence (excitation: 295 nm; emission: 340 nm). Asparagine was not measurable because its peaks were too close to those of NBD-F derivatives. Because the minimum volume required for all our assays (NBD-F labeling, SBD-F labeling, and Trp detection) is 8 µL (the routine serum volume used was 25 µL), equal CSF volumes (3–5 µL) of two–three mice were pooled. For the detection of thiol-containing amino acids, serum and CSF samples were reduced to cleave disulfide bonds before SBD-F labeling, and the total levels of Hcy, Cys, and GSH (tHcy, tCys, and tGSH, respectively) were measured [28 (link),29 (link)].
4
NBD-F Labeling of Phospholipids
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Chemical modification of the amino group of phospholipids with 4-fluoro-7-nitrobenzofurazan (NBD-F; Dojindo) was performed according to the manufacturer’s protocol. E. coli total phospholipids in chloroform (Avanti Polar Lipids) were dried up thoroughly by nitrogen gas to form thin films, which were then hydrated by 50 mM boric acid buffer, pH 8.0. The hydrated phospholipids were modified by NBD-F at 65°C for 1 min. The reaction was stopped by cooling on ice. The labeled phospholipids, including crude NBD-PE, were extracted according to the methods reported previously (Ichihara et al., 2011 (link)). In brief, methyl tert-butyl ether and methanol were added to the sample (2:1:2 vol/vol) and subjected to a low-speed centrifuge. The resulting upper layer was transferred to new test tubes, dried by a nitrogen stream, and then dissolved in TBS300 to be hydrated. The hydrated crude NBD-PE was mixed with the cell extracts containing free Mdm12 or Mmm1s-Mdm12. Proteins loaded with NBD-PE were purified by the protein purification procedures described in the Protein expression, purification, and modification section.
5
Amino Acid Analysis in Cbs and Cth Mice
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Heterozygous Cbs +/-mice 9) (link) were obtained from the Jackson Laboratory (Bar Harbor, ME, U.S.A.) and Cth +/-mice were generated by our group. 8) (link) Heterozygous mice on a C57BL/6J background were bred to obtain Cbs -/-or Cth -/-mice. Blood and CSF samples were collected from 2-week-old pups before Cbs -/-mice start to die. 7, (link)9) (link) Mice were anesthetized using isoflurane; serum and CSF samples were collected by cardiocentesis (ca. 15 µL per pup) and ventricle puncture with glass capillaries (2-4 µL per pup), respectively. Serum/CSF amino acid levels were measured using HPLC, amino acid labeling with 4-fluoro-7-nitrobenzofurazan (NBD-F [Dojindo, Japan]), and thiol labeling with 4-fluoro-7-sulfobenzofurazan (SBD-F [Dojindo]) as described previously. 7, (link)8, (link)10) (link) Because 10 µL is the minimum volume required for our assays, equal volumes of CSF from 4-6 mice were pooled together. Aspartic acid/asparagine (Asp/Asn) peaks were indistinguishable and tryptophan (Trp) was not derivatized with NBD-F. For the detection of thiol-containing amino acids, serum/CSF samples were reduced to cleave disulfide bonds before SBD-F labeling, and the total levels of homocysteine, cysteine, and glutathione (tHcy, tCys, and tGSH, respectively) were measured.
6
NBD-F Labeling Reagent Preparation
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NBD-F was obtained from Dojindo Laboratories (Kumamoto, Japan). Amino acids, tetrapentylammonium bromide (HPLC grade), and tetraheptylammonium bromide (HPLC grade) were purchased from Sigma-Aldrich (St. Louis, MO). Tetrabutylammonium bromide (HPLC grade) was acquired from Nacalai Tesque (Kyoto, Japan). Methylamine was obtained from Wako Pure Chemical (Osaka, Japan). Acetonitrile (HPLC grade) was purchased from Merck (Darmstadt, Germany). Water was purified using a Milli-Q system from Merck Millipore (Darmstadt, Germany).
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