Reaction #543550

Reaction equation

Tris-HCl

CoA

sinapinic acid

→

crotonyl-CoA

Solvents

TFA

acetonitrile

Conditions

Workup

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

Procedure

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.

Source