Реакция #1178398

Уравнение реакции

NaCl

UDP-GalNAc

→

GalNAc

Растворители

water

Условия реакции

Обработка

1. 1
   Другоеobtained by chemical desulfation of CS
2. 2
   Другоеa K4CP enzyme solution (recombinant enzyme obtained
3. 3
   workup.ADDITIONcorresponding to 37.5 μg protein) was added
4. 4
   workup.WAITthermal inactivation was performed for 1 minute
5. 5
   Другоеthe reaction mixture was subjected to ethanol precipitation
6. 6
   workup.DISSOLUTIONthe residue was dissolved again in 200 μl of distilled water
7. 7
   ФильтрацияThe solution was subjected to gel filtration chromatography in a Superdex 75 HR10/30 column
8. 8
   Другоеwere collected every 1 minute (1 ml)
9. 9
   workup.DISSOLUTIONeach of the lyophilized fractions was dissolved again in 10 μl of distilled water
10. 10
    ДругоеFrom a part of the solution, 10 μl of a 1 nmole/μl solution was prepared
11. 11
    Другоеto prepare a sample for MALDI-TOF-MS

Методика

To an enzymatic reaction mixture (500 μl) containing CH obtained by chemical desulfation of CS and having different non-reducing ends (1 mg, average molecular weight: 10,000, Seikagaku Corporation), UDP-GalNAc (3 μmol) 50 mM Tris-HCl buffer, pH 7.2, 20 mM MnCl2 and 0.15 M NaCl, a K4CP enzyme solution (recombinant enzyme obtained according to the procedure of the example of Japanese Patent Laid-open Publication (KOKAI) No. 2003-199583; corresponding to 37.5 μg protein) was added. A reaction was performed at 30° C. for 18 hours, thermal inactivation was performed for 1 minute in boiling water, the reaction mixture was subjected to ethanol precipitation, and the residue was dissolved again in 200 μl of distilled water. The solution was subjected to gel filtration chromatography in a Superdex 75 HR10/30 column using 0.2 M ammonium acetate as a developing buffer. The solution was loaded at a flow rate of 1 ml/minute, and fractions were collected every 1 minute (1 ml). The obtained fractions were lyophilized, and each of the lyophilized fractions was dissolved again in 10 μl of distilled water. From a part of the solution, 10 μl of a 1 nmole/μl solution was prepared, and the solution was loaded on a small amount of Dowex 50 XW8 (H+ form) gel to prepare a sample for MALDI-TOF-MS.

Источник