Reacción #833546

Ecuación de reacción

Tris-HCl

CoA

sinapinic acid

→

crotonyl-CoA

Disolventes

TFA

acetonitrile

Condiciones de reacción

Tratamiento posterior

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

Procedimiento

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% trifluroacetic 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.

Fuente