Soluble chromatin was released from the nuclei under physiological conditions and was fractionated on a 6 40% isokinetic sucrose gradient in TEEP80 buffer.
The NaOH was neutralized by the addition of 5 µL of 4 M acetic acid and after the addition of Laemmli buffer proteins were fractionated by SDS polyacrylamide gel electrophoresis and analyzed by immunoblotting using anti-HSF1 (Neef et al. 2010) and anti-PGK1 (22C5D8; Abcam) antibodies.
Samples containing 50 75 µg of protein in reducing sample buffer were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis.
After selection for 10 14 days, the cell lysates prepared from the pooled population of cells in the sampling buffer were fractionated on SDS-PAGE for immunoblotting detection of Bmi-1 protein level.
Western blots were carried out by lysing transfected cells (30 μl) in 1× Laemmli SDS-PAGE loading buffer [ 68] and proteins were fractionated on a 10% SDS-PAGE gel with a constant current of 35 mA.
For in-gel analyses, reaction products from in vitro ubiquitin conjugation assays, halted by the addition of SDS sample buffer containing reducing agent, were fractionated by SDS/PAGE (Novex NuPAGE 10% Bis-Tris gel; Life Technologies) using Mes SDS running buffer.
mAbs and their fragments were fractionated by 12.5% SDS PAGE.
Beads were then washed 5 times with IP lysis buffer and immunoprecipitates were eluted in Laemmli buffer at 99 °C for 10 min. For immunoprecipitation experiments of mitochondrial and cytosolic fractions, the cells were fractionated in advance as described below.
Samples stored in RNAlater were fractionated and lyzed by adding 10 × volume (v/v) lysis buffer.
