After centrifugation in a Beckman SW28 rotor (27.000 rpm, 4°C, 3 h), pelleted nuclei were washed twice with cold TES, precipitated in a Beckman JA-25.50 JA-25.50.000 rotor10 min, 4°C) and resuspended in the same buffer.
Integral and peripheral protein fractions were resuspended in PBS, cold acetone precipitated, centrifuged and resuspended in washing buffer containing also the Complete Mini EDTA-free Protease Inhibitor Coctail (Roche).
After incubation for another 22-24 hours at 4°C, 10 μl of rabbit carrier and 1.0 ml of cold (4°C) precipitating reagent were added and incubated for 20 minutes at 4°C.
The intrinsic performance characteristics of this novel method include its sensitivity and specificity, even with thick and sticky cold-precipitating samples.
The relaxed "cold" DNA is precipitated as described above and resuspended in a total of 500 μl H2O.
After washing in TFA, filtrate was collected and added into cold ether and precipitated peptides were produced; crude peptides were then collected and vacuum dried.
The cells were washed twice with ice cold PBS, followed by precipitated with ice-cold 5% TCA twice, each with 10 minutes.
Cells were then washed twice with 500 µl of chilled HBSS, fixed with 250 µl of ice-cold methanol and precipitated by 250 µl of 10% trichloroacetic acid.
Protein pellets were washed twice with ice-cold acetone, and precipitated via centrifugation, as previously.
Sequencing products were ethanol precipitated in cold 95% ethanol, washed twice with cold 70% ethanol, dried for 20 minutes, and then re-dissolved in sample loading solution provided by the manufacturer (Beckman Coulter, Fullerton, CA).
Briefly, cells were trypsinized, washed with cold PBS, and then fixed with ice-cold 70% ethanol and precipitated for 2 h at ice.
