Pierce 660 protein assay reagent

Cells were harvested by centrifugation into a pellet 48 h post-transfection, resuspended in ice-cold lysis buffer (PBX supplemented with 1% Triton-X, 150 mM NaCl, and Roche cOmplete protease inhibitor #11836170001; Indianapolis, IN, USA), and sonicated for 15 s at 30% power (FisherBrand #FB50110; Waltham, MA, USA). Lysates were centrifuged for 15 min at 12,000 rpm at +4 °C, whereupon the supernatant (total lysate) was transferred to a fresh tube. Then, 150 μg of the total lysate, as determined by the Pierce 660 Protein Assay Reagent (ThermoFisher #22660; Waltham, MA, USA), was subjected to Co-IP for a 2 h rotation at +4 °C in a total volume of 500 μL. For Western blot, 1 μg of an anti-GFP antibody (Santa Cruz; #sc-9996; Dallas, TX, USA) and 10 μL of Dynabeads Protein G (ThermoFisher #10004D) were mixed with the total lysate. Samples were then washed three times for 10 min before elution in SDS sample buffer by heating in a 37 °C water bath for 15 min.

Cells were seeded in 75 cm² culture flask (Corning, #430641U), rinsed once with PBS and lysed with radioimmunoprecipitation assay buffer (20 mM tris hydroxymethyl, 150 mM sodium chloride, 1 mM EDTA, 1 mM EGTA, 1% sodium deoxycholate, 10% glycerol, 1% NP40, pH 7.6) supplemented with protease inhibitor cocktail (Sigma-Aldrich, 11697498001) and phosphatase inhibitor buffer (25 mM sodium orthovanadate, 250 mM 4-nitrophenylphosphate, 250 mM ß-glycerophosphate, 125 mM sodium fluoride). Lysates were sonicated 3 x 10 sec (amplitude 50) and centrifuged (10 min, 15000 g). Cleared cell lysates were assessed for protein content with Pierce 660 Protein Assay Reagent (ThermoFisher, 22660) according to the manufacturer′s recommendations.

This was done based on a published protocol^{76 (link)}. Briefly, overnight cultures of wt, swi1∆ and [SWI⁺] cells in liquid YPD were properly diluted into the same medium and harvested at log and stationary phases. Cell pellets were collected after centrifugation at 660 g for 3 min and washed twice with water. Cell densities were determined after cell counting. Cells were disrupted by glass beads for 6 × 1 min using a bead-beater in lysis buffer (50 mM sodium phosphate buffer, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol, 2 mM PMSF, 2 μg/mL pepstatin A, 2 μg/mL leupeptin, 5 μg/mL aprotinin and 1 mM benzamidine-HCl). Lysates were centrifuged at 500 g for 5 min at 4 °C to remove cell debris. Supernatants were transferred to new tubes and the protein concentration was determined using Pierce 660 protein assay reagent (Thermo Scientific, ProD# 22,660) based on a protocol provided by the manufacturer. Different concentrations of BSA were used as control to set the standard of protein concentrations. Multiple tests were performed with at least three bio-repeats in each test. Protein concentrations were then normalized to cell numbers as outcomes.