Pierce coomassie plus protein assay reagent

Cells were lysed in lysis buffer (150 mM NaCl, 50 mM Tris HCl, 5 mM EDTA, 1% NP40, 3 mM PMSF, 1/100 protease inhibitor cocktail III [Calbiochem 539134] and 1/100 phosphatase inhibitor cocktail II [Calbiochem 524625]) on ice for 5 min and centrifuged at 20,000g for 30 min. The proteins in the supernatant were removed and quantified using a Pierce Coomassie plus protein assay reagent (23236, Thermo Fisher Scientific). For western blot analyses, proteins were separated on 7.5% Mini-PROTEAN TGX™ precast protein gels (456-1026, Biorad), transferred to PVDF membranes (Millipore) and probed with either an anti-PAR rabbit polyclonal antibody (4336-BPC-100, Trevigen), an anti-phospho-Chk1 (Ser345) rabbit monoclonal antibody (2348, Cell signalling), an anti-phospho-p53 (Ser15) mouse monoclonal antibody (9286, Cell signalling), an anti phospho-Chk2 (Thr68) rabbit polyclonal antibody (2661, Cell signalling) or an anti-actin rabbit polyclonal antibody (A2066, Sigma-Aldrich).

Protein concentration in the extracts was determined by a standard spectrophotometer (595 nm) following the procedures described by Bradford [54 (link)], using the Pierce™ Coomassie Plus™ Protein Assay Reagent (Thermo Fisher Scientific, Waltham, Massachusetts, United States) and bovine serum albumin (BSA) as a standard.

Cells were lysed on ice using RIPA buffer (50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 1% triton X-100, 0.1% sodium deoxycholate and 0.1% SDS) supplemented with protease and phosphatase inhibitor cocktails (Roche). Protein extracts were clarified and concentrations were measured with Pierce Coomassie Plus Protein Assay reagent (Thermo Fisher Scientific). Lysates were then resolved on SDS-PAGE gels (BioRad), and transferred to Nitrocellulose blots. Membranes were probed with primary antibodies against AXIN1 (2087, CST), TCF4 (2569, CST) pFAK Y397 (SAB4504403, Sigma), DUSP6 (EPR129Y, Abcam), SPRY2 (EPR4318(2)(B), Abcam), pERK (4370, CST), tERK (4696, CST), Vinculin (EPR8185, Abcam), and β-tubulin (E7, DSHB). Blots were subsequently incubated with HRP conjugated secondary antibodies. For signal development, Supersignal West Femto Substrate kit (Thermo Fisher Scientific) was used, followed by image acquisition using darkroom development. ImageJ was used for band intensity quantitation.