Reaktion #2028917

ord-d11127dce50a41d89a5cb6d853e160cd

Reaktionsgleichung

O=C1C=Cc2n/c(=C3\N[C@@H](C(=O)O)CS3)sc2=C1
luciferin
O=S(=O)([O-])CCS.[Na+]
MESNA
Nc1ncnc2c1ncn2[C@@H]1O[C@H](COP(=O)(O)OP(=O)(O)O)[C@@H](O)[C@H]1O
ADP
O=C(O)CN(CCN(CC(=O)O)CC(=O)O)CC(=O)O
EDTA
→
Nc1ncnc2c1ncn2[C@@H]1O[C@H](COP(=O)(O)O)[C@@H](O)[C@H]1O
Adenylate

Edukte

O=C1C=Cc2n/c(=C3\N[C@@H](C(=O)O)CS3)sc2=C1
luciferin
O=S(=O)([O-])CCS.[Na+]
MESNA
Nc1ncnc2c1ncn2[C@@H]1O[C@H](COP(=O)(O)OP(=O)(O)O)[C@@H](O)[C@H]1O
ADP
O=C(O)CN(CCN(CC(=O)O)CC(=O)O)CC(=O)O
EDTA

Reagenzien

Keine

Lösungsmittel

NC(CO)(CO)CO
Tris

Reaktionsbedingungen

Detaillierte Bedingungen
See reaction.notes.procedure_details.

Aufarbeitung

  1. 1
    SonstigeStandard curves for the AK enzyme was prepared
  2. 2
    workup.ADDITIONadded to each well
  3. 3
    SonstigeThree separate standard curves
  4. 4
    Sonstigewere prepared for incubation of the assay plate at 30 degrees C
  5. 5
    Sonstige50 degrees C
  6. 6
    Sonstige70 degrees C
  7. 7
    workup.ADDITIONwas added

Vorschrift

Standard curves for the AK enzyme was prepared as follows. Serial dilutions of purified Sulfolobus acidocaldarius AK from 10 microgrammes/ml to 1 fg/ml were prepared in 50 mM Tris, 25 mM MESNA, pH 7.3. 100 microlitres of enzyme was added to each well of a microtitre plate and 100 microlitres of 135 micromolar ADP 15 mM MgAc, 1 mM EDTA added to each well. Three separate standard curves were prepared for incubation of the assay plate at 30 degrees C., 50 degrees C. and 70 degrees C. for 20 minutes. Following incubation 30 microlitres of luciferin/luciferase reagent (Biothema) was added and the signal read in a luminometer (Orion, Berthold) and the results shown in FIG. 3.

Quelle

DOI: 10.6084/m9.figshare.5104873.v1Patent: US08389208B2uspto-grants-2013_03