Réaction #1552063

Équation de réaction

Tris-HCl

CoA

sinapinic acid

→

crotonyl-CoA

Solvants

TFA

acetonitrile

Conditions de réaction

Traitement

1. 1
   Extractionfree extract
2. 2
   Autreto proceed at room temperature
3. 3
   workup.ADDITIONby adding 1 volume 10% trifluoroacetic acid (TFA)
4. 4
   AutreThe reaction mixtures
5. 5
   Autrewere purified
6. 6
   LavageThe column was washed twice with 1 mL 0.1% TFA
7. 7
   LavageThe sample was eluted in 200 μL 40% acetonitrile
8. 8
   AutreThe acetonitrile was removed by centrifugation in vacuo
9. 9
   AutreSamples were prepared for MALDI-TOF MS analysis
10. 10
    Autreto air dry

Mode opératoire

The assay mixture contained 50 mM Tris-HCl (pH 7.5), 1 mM CoA ester, and about 1 μg cell free extract. Reactions were allowed to proceed at room temperature and were stopped by adding 1 volume 10% trifluoroacetic acid (TFA). The reaction mixtures were purified prior to MALDI-TOF MS analysis using Sep Pak Vac C18 50 mg columns (Waters, Inc.). The columns were conditioned with 1 mL methanol and then equilibrated with two washes of 1 mL 0.1% TFA. The sample was applied to the column, and the flow through was discarded. The column was washed twice with 1 mL 0.1% TFA. The sample was eluted in 200 μL 40% acetonitrile, 0.1% TFA. The acetonitrile was removed by centrifugation in vacuo. Samples were prepared for MALDI-TOF MS analysis by mixing 1:1 with 110 mM sinapinic acid in 0.1% TFA, 67% acetonitrile. The samples were allowed to air dry.

Source