反応 #2028917

ord-d11127dce50a41d89a5cb6d853e160cd

反応方程式

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

反応物

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

試薬

なし

溶媒

NC(CO)(CO)CO
Tris

反応条件

詳細条件
See reaction.notes.procedure_details.

後処理

  1. 1
    その他Standard curves for the AK enzyme was prepared
  2. 2
    workup.ADDITIONadded to each well
  3. 3
    その他Three separate standard curves
  4. 4
    その他were prepared for incubation of the assay plate at 30 degrees C
  5. 5
    その他50 degrees C
  6. 6
    その他70 degrees C
  7. 7
    workup.ADDITIONwas added

実験手順

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.

出典

DOI: 10.6084/m9.figshare.5104873.v1特許: US08389208B2uspto-grants-2013_03