For all pNPs except for pAc, a 1 mM pNP solutions was prepared in 50 mM sodium acetate buffer pH 5, and distributed into polystyrene 96-well plates (IWAKI, Japan) at 100 μl per well and one column per substrate.
Subsequently, this structure was dissected and homogenized in 20 40 volumes of cold 150 mM potassium phosphate buffer, pH 6.8, and distributed into two aliquots, one to be used for the analysis of protein concentration.
A 1% w/v suspension/solution was prepared in 50 mM sodium acetate buffer, pH 5, and distributed into 96-well plates at 100 μl per well and one column per substrate.
Bisulfite-treated DNA was eluted in 10 μl of elution buffer, diluted to a final volume of 200 μl and evenly distributed into 20 wells of a 96-well plate.
After the washings the packed ghosts were suspended in 2.6 ml buffer A and this suspension is distributed into four tubes according to the following scheme: Ghosts and buffer were mixed gently and the glasses were placed at appropriate temperature with gentle shaking (see above).
The proposed method uses the Loop-back Buffer adaptively and distributes traffics in time and space domain.
Briefly, TS and XEN cells were sorted by fluorescence-activated cell sorting using the MoFlo system (Beckman Coulter, Brea, CA, USA), and individual cells were distributed into wells or 96-well plates containing 5 μl of CellsDirect resuspension buffer (Invitrogen, Carlsbad, CA, USA).
Briefly, TS cells were sorted by fluorescence-activated cell sorting using the MoFlo system (Beckman Coulter, Brea, CA, USA), and individual cells were distributed into wells or 96-well plates containing 5 μl of CellsDirect resuspension buffer (Invitrogen, Carlsbad, CA, USA).
25 µl each of lysate from a single well was distributed into three wells and firefly and renilla luciferase activities were sequentially measured with L-Max luminometer which injected 100 µl each of LAR II buffer and Stop&Gro buffer (Molecular Devices, Sunnyvale, CA).
