反应 #833546

反应方程式

Tris-HCl

CoA

sinapinic acid

→

crotonyl-CoA

溶剂

TFA

acetonitrile

反应条件

后处理

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

实验过程

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.

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