At mole ratio > 0.6, the incorporation started to decline, possibly caused by the presence of other cations (derived from decay product of 213Bi, quencher, buffer, elution solution) in the labelling reaction which interfered with the incorporation of 213Bi into DOTA-moiety (Breeman et al. 2005; Zhernosekov et al. 2007).
The pellets were air-dried and resuspended in dissolution buffer provided in the iTRAQ labelling kit (Applied Biosystems).
The amplifier (2 nmol/L) was in hybridization buffer 2. The label probe (2 nmol/L) was in hybridization buffer 3 (5× SSC, 0.3% lithium dodecyl sulfate, blocking reagents) [ 10].
The H3 protein was incubated with MLL3-SET in methylation buffer in the presence of either radioactively labeled or unlabeled AdoMet for 3 h at 25°C.
After two washes in phosphate-free Krebs buffer, the labeled parasites were lysed for 30 min at 4°C in lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40) supplemented with protease and phosphatase inhibitors (Roche), the resulting lysate was centrifuged at 20,000 g for 5 min and the supernatant collected.
The labeling of Dpo4 mutants was performed in the labeling buffer [50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 10% glycerol, and 0.5 mM TCEP].
Briefly, after trypsin release, 10 × 10 cells were washed once in basic medium and resuspended into 1 ml of dilution buffer provided by the manufacturer in the labeling kit.
The slides were washed with 1 mL of the wash buffer provided in the kit and labeled with the DNA labeling solution at 37°C for 60 minutes.
A 50-μm diameter N-CHO coated capillary with separation length ~ 42 cm and carbohydrate separation buffer (both included in the carbohydrate labeling and analysis kit) were used for separating amylopectin branches.
