Anykd sds page gel

Proteins were selected for validation by western blot analysis based on significance as well as function. Proteins were separated on an AnyKD SDS-PAGE gel (BioRad) and transferred to a PVDF membrane using the Trans Turboblot system (BioRad). Membranes were blocked in 5% non-fat milk in TBS for 1 hour at room temperature. Primary antibodies specific for phospho-Serine47-Histone H4 (rabbit polyclonal, 1:500), Histone H4 (mouse monoclonal, 1:1000), PCTAIRE-2 and PCTAIRE-3 (rabbit polyclonal, 1:1000), actin (mouse monoclonal, 1:7000) and GAPDH (rabbit monoclonal, 1:5000) were diluted in 5% BSA-TBS, containing 0.05% sodium azide and incubated overnight at 4°C. Membranes were washed and then incubated with appropriate corresponding secondary antibodies, donkey anti-rabbit HRP-conjugated or goat anti-mouse HRP-conjugated for 1.5 hours at room temperature. After further washes, all blots were developed with Pico Chemiluminescence reagents, with the exception of pS47-Histone H4 which was developed using Femto Chemiluminescence reagents, using an Amersham Imager 600RGB (GE Healthcare).

After 48 h transfection, cells were trypsinized and washed three times with PBS and then lysed in lysis buffer for 30 min on ice. The protein was then electrophoresed on an AnyKD SDS-PAGE gel (Bio-Rad, Hercules, CA, USA) and then transferred to a nitrocellulose membrane (Bio-Rad) and blocked with 5% non-fat milk in TBST for 2 h at room temperature with shaking. The membrane was immunoblotted with primary antibody for rabbit anti-GAPDH, anti-MITF and anti-MAP4K3 (Cell Signaling Technology, Danvers, MA, USA) with dilutions of 1 : 1000, 1 : 1000 and 1 : 1000, respectively, at 4 °C overnight. Signals were developed using HRP-linked secondary antibody (1 : 10 000) with Clarity Western ECL Substrate (Bio-Rad). The intensity of the signals was determined by FluorChem E system (Protein Simple, Santa Clara, CA, USA).

The flow through and wash fraction from the affinity chromatography were pooled (22 ml) and concentrated to 10 ml using a 15 ml Amicon centrifugal cellulose membrane filter (10 kDa MWCO). The concentrated sample was passed through a 0.2 μm filter before loading it using a 10 ml superloop onto a 320 ml HiLoad 26/600 Superdex 75 pg gel filtration column (GE Healthcare) connected to an ÄKTA Purifier system. The column was run at 4 °C, with a flow rate of 2.6 ml/min. 100 mM NH4OAc pH 6.0, 150 mM NaCl was used as running buffer and 5 ml fractions were collected. Samples from the collected fractions were analyzed on a Criterion TGX Precast Any kD SDS-PAGE gel (Bio-Rad) stained with Bio-Safe Coomassie (Bio-Rad). The concentration Bcl3-ARD in the three different pools from the size exclusion chromatography was measured in a NanoDrop spectrophotometer.