The 3Cpro, the best fluorogenic peptide and compound incubate a few minutes in buffer.
The loading procedure was performed twice before the vessels were washed and equilibrated for 15 minutes in buffer solution.
In brief, tissue sections were subjected to pre-wash twice for 5 minutes in buffers, followed by incubating with [3H]gaboxadol: 10–400 nM and post-wash with buffers for 4 quick dips.
For reactions with D-loops containing a biotinylated nucleotide the substrate was pre-incubated at 37°C for 10 minutes in buffer alone or with 1.5 nM streptavidin prior to WRN addition.
Ferricyanide reductase assays were conducted at room temperature and the absorbance monitored at 410 nm for 2 minutes in buffer containing 10 mM potassium phosphate (pH 7.0), 1 mM EDTA, 1 mM K3FeCN6, and 10 mM KCN [46].
The slides were then left to stand for 10 minutes in buffer at room temperature before being washed thoroughly in tap water.
Protein samples were boiled for 2 minutes in buffer containing: 100 mM Tris-HCl (pH 6.8), 240 mM β-mercaptoethanol, 4.38 mM SDS, 3 μM bromophenol blue, and 20% glycerol to a final total protein concentration of 0.125 mg/ml.
Nuclear pellets were harvested after centrifugation at 10,000 rpm for 5 minutes at 4°C, and lysed for 20 minutes in buffer containing 20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 420 mM NaCl, 25% glycerol, 1 mM PMSF and 5 mM DTT.
Slides with fresh frozen tissue were post-fixed with 100% ethanol for 10 minutes, followed by 3 washes for 10 minutes in phosphate buffered saline (PBS).
