Thermoscientific pierce bca protein assay

Cells were lysed in 1% 3-[(3-cholamidopropyl) dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPS) buffer (50 mM HEPES, 100 mM NaCl, pH 7.4 and 1% CHAPS) supplemented with HALT protease and phosphatase inhibitor cocktails (ThermoScientific, Waltham, MA) by pipetting up-and-down, passing through a 37-gauge needle and rotating for 1 h at 4 °C. The lysates were centrifuged at 14,000xg for 15 min and the supernatants were collected for the IP. Total protein in the samples was measured using ThermoScientific Pierce BCA Protein Assay (ThermoScientific, Waltham, MA). The aliquots of the supernatant containing equal protein amount were incubated with 3 μg of the respective antibody or normal IgG, as a negative control, with end-over-end rotation over night at 4 °C. Subsequently, 30 μl of Protein G Dynabeads (ThermoScientific, Waltham, MA) were added to the samples and incubated with end-over-end rotation for 10 min at room temperature. The beads were collected using a magnetic tube rack, washed twice with the 1% CHAPS buffer and once with the wash buffer containing 50 mM HEPES, 100 mM NaCl, pH7.4. The protein was eluted by 5-min boiling in 25 μl 2x LDS, 1x reducing agent buffer (ThermoScientific, Waltham, MA).

Cells were harvested and lysed in the lysis buffer (1.25 M urea and 2.5% SDS) after washing with phosphate-buffered saline (PBS). The viscosity of the lysate was removed by sonication, and protein concentration measured by Thermo Scientific Pierce BCA Protein Assay (Thermo Fisher Scientific, #PI-23227). Primary antibodies performed are listed in the Supplementary Table 2. WB detection was performed using chemiluminescent detection reagents (Bio-Rad #170-5061 or Thermo Scientific #34075) and ImageQuant LAS 4010 (GE Healthcare).

Cells were lysed in radio-immunoprecipitation assay (RIPA: 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% sodium dodecyl sulphate [SDS], 1% Triton X-100, 1% sodium deoxycholate, 5 mM EDTA, 1 mM NaF, 1 mM sodium orthovanadate and protease inhibitors) buffer as we previously described [16 (link)]. Clarification of lysates occurred through centrifugation at 15,000 g for 10 min, and the level of protein concentration was quantified using the bicinchoninic acid assay kit (Thermo Scientific™ Pierce™ BCA Protein Assay, Thermo Fisher Scientific, Waltham, MA). Proteins (10–30 μg) were resolved by SDS-PAGE and then transferred on polyvinylidene fluoride (PVDF) membrane. Immunoblotting of lysates was performed with primary antibodies (Table 1), and following incubation with secondary antibodies, proteins were detected using Luminata™ Western Chemiluminescent HRP Substrate (Millipore-Sigma). Immunoreactive bands were quantified based on pixel intensity using FluorChem Q Imaging software (Alpha Innotech Corp, San Leandro, CA). Data is represented as being either normalized to the respective unphosphorylated form or to β-actin as loading controls for each respective condition.