反応 #1960510

反応方程式

AEBSF

glycerol

Hepes

→

Lysine

反応条件

後処理

1. 1
   その他The cells then underwent continuous sonication for 10 minutes at 4° C.
2. 2
   workup.WAITThe total cell lysate was centrifuged at 4° C. at 20000 g for 15 min
3. 3
   workup.ADDITIONcontaining 1 mg of protein per ml
4. 4
   その他purified on a heparin column to 0.5 mg/ml, 5 μl of 1M MgCl2 and 55 μl of buffer A
5. 5
   その他were carried out at 25° C
6. 6
   その他100 μl samples were removed at intervals

実験手順

smegmatis or M. bovis BCG cultures with an optical density measured at 600 nm (OD600) of 0.5 were centrifuged at 10000 g for 15 min. The pellet was re-suspended in 10 ml of 50 mM Hepes buffer (pH 7.4) containing 1 mM of AEBSF (Pefabloc Sc, Roche) and 15% (v/v) of glycerol (buffer A). The cells then underwent continuous sonication for 10 minutes at 4° C. using a Branson type sonicator, the outlet power being adjusted to 5. The total cell lysate was centrifuged at 4° C. at 20000 g for 15 min. For the methylation tests, 300 μl of total clarified lysate containing 1 mg of protein per ml was mixed with 40 μl of [methyl-14C]AdoMet (60 mCi/mmol, Amersham Pharmacia Biotech), 100 μl of recombinant HBHA purified on a heparin column to 0.5 mg/ml, 5 μl of 1M MgCl2 and 55 μl of buffer A. The methylation tests were carried out at 25° C. 100 μl samples were removed at intervals to verify the degree of methylation of the recombinant HBHA by autoradiography.

出典