반응 #529801
ord-95c734f0097b4bf79f50b4b3023ee2a5
반응 조건
후처리
- 1기타Briefly, reaction constituents (all from Sigma chemicals)
- 2기타were prepared
- 3workup.ADDITIONadded to a microcentrifuge tube
- 4기타was placed into a 37° C.
- 5기타dry block
- 6추출Next, 100 μl of diluted cell-free extract (dilutions range between 1:2 and 1:50)
- 7workup.ADDITIONwas added
- 8workup.ADDITIONthe contents mixed
- 9workup.WAITto proceed for 10 minutes
- 10workup.ADDITIONby adding 450 μl of ferric chloride reagent (see below)
- 11workup.WAITthe tube was placed on ice for 10 min
- 12workup.WAITThe tube was then centrifuged in a microcentrifuge for 2 min
- 13workup.ADDITIONa blank containing all of the above constituents except for 100 μl of deionized water
- 14기타in place of the enzyme preparation
실험 절차
Acetate kinase levels were measured essentially according to the method described by Brown et al., Journal of General Microbiology 102:327-336; 1977. Briefly, reaction constituents (all from Sigma chemicals) were prepared, and added to a microcentrifuge tube as follows: 12.5 μl of 200 mM magnesium chloride, 50 μl of 100 mM ATP, 30 μl of 200 mM sodium acetate, 50 μl of hydroxylamine solution (see below), and 30 μl of water. All of these solutions except for magnesium chloride are made in 50 mM Tris buffer, pH 8.0. The tube was placed into a 37° C. dry block and equilibrated for 3 minutes. Next, 100 μl of diluted cell-free extract (dilutions range between 1:2 and 1:50) was added, the contents mixed, and the reaction allowed to proceed for 10 minutes. The reaction was stopped by adding 450 μl of ferric chloride reagent (see below), and the tube was placed on ice for 10 min. The tube was then centrifuged in a microcentrifuge for 2 min, and absorbance at 540 nanometers determined versus a blank containing all of the above constituents except for 100 μl of deionized water in place of the enzyme preparation.