2-Mercaptoethanol → Resazurin

HRID 333577

Reaktionsgleichung

Reaktionsgleichung von HRID 333577
[Na+].[Cl-].[Mg+2].[Cl-].[Cl-].C(O)C(N)(CO)CO.SCCO.C1N=C(N)C2N=CN([C@@H]3O[C@H](COP(OP(OC[C@H]4O[C@@H](N5C=C(C(N)=O)CC=C5)[C@H](O)[C@@H]4O)(O)=O)(O)=O)[C@@H](O)[C@H]3OP(O)(O)=O)C=2N=1.[CH:66]1[C:71]([OH:72])=[CH:70][C:69]2[O:73][C:74]3[C:80](=[N+:81]([O-:82])[C:68]=2[CH:67]=1)[CH:79]=[CH:78][C:76](=[O:77])[CH:75]=3>CS(C)=O>[CH:78]1[C:76]([OH:77])=[CH:75][C:74]2[O:73][C:69]3[C:68](=[N+:81]([O-:82])[C:80]=2[CH:79]=1)[CH:67]=[CH:66][C:71](=[O:72])[CH:70]=3.[CH:78]1[C:76]([OH:77])=[CH:75][C:74]2[O:73][C:69]3[C:68](=[N:81][C:80]=2[CH:79]=1)[CH:67]=[CH:66][C:71](=[O:72])[CH:70]=3

Komponenten

9

Edukt 6

HCID 15672-MercaptoethanolHCID 5234Sodium ChlorideHCID 5884NADPHHCID 6503Tris(hydroxymethyl)aminomethaneHCID 11077ResazurinHCID 5360315Magnesium Chloride

Produkt 2

HCID 11077ResazurinHCID 69462Resorufin

Reagenzien, Katalysatoren, Lösungsmittel 1

HCID 679Dimethyl SulfoxideLösungsmittel

Versuchsvorschrift

Assays were conducted in a standard 384-well plate in a reaction volume of 76 uL assay buffer (150 mM NaCl, 10 mM MgCl2, 20 mM Tris pH 7.5, 0.05% bovine serum albumin, 2 mM beta-mercaptoethanol) as follows: To 25 uL of substrate mix (4 uM NADPH, 1 mM aKG), 1 uL of test compound in DMSO was added. The plate was centrifuged briefly, and then 25 ul of enzyme mix was added (0.1 ug/mL ICDH1 G97D) followed by a brief centrifugation and shake at 100 RPM. The reaction was incubated for 50 minutes at room temperature, then 25 ul of detection mix (30 uM resazurin, 36 ug/ml diaphorase) was added, and the mixture further incubated for 5 minutes at room temperature. The activity readout as a result of the conversion of resazurin to resorufin was detected by fluorescent spectroscopy at excitation 544 nm and emission 590 nm (c/o 590 nm).

Aufarbeitung

  1. additionwas added
  2. addition25 ul of enzyme mix
  3. additionwas added (0.1 ug/mL ICDH1 G97D)
  4. additionwas added
  5. waitthe mixture further incubated for 5 minutes at room temperature

Quellen und Nachweise

ORD-Eintrag
ord-f8457f81ae4e465baaf3137ffd382180
Quelldatensatz
uspto-grants-2014_11
Patentnummer
US08883438B2

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