Bicinchoninic acid working reagent

PEA and PMA polymers were synthesized by radical polymerization of ethyl acrylate and methyl acrylate (Sigma, St. Louis, MO), initiated by benzoin at 1 wt% and 0.35 wt%, respectively. PEA and PMA were then dried by vacuum extraction to constant weight and solubilized in toluene 2.5% w/v and 6% w/v respectively. PEA and PMA films (~1μm) were obtained by spin-casting polymer solutions on glass coverslips (12 or 25mm) at 2000 (PEA) and 3000 (PMA) rpm for 30 seconds, with an acceleration of 3000 rpm/sec. Samples were dried in vacuum for 2 h at 60^oC to remove excess toluene. Polymer surfaces were coated with either natural mouse laminin 111 (LM; Invitrogen) or human plasma FN (Sigma) solutions, in Dulbecco’s phosphate-buffered saline (DPBS) at concentrations 2, 10, 20, 50, and 100 μg/mL for 1h and then rinsed with DPBS before use. Mouse plasma fibronectins carrying mutations in the FNIII₁₀ module were also used. ΔRGD FN (FN without the RGD sequence) and syn FN (FN with a mutation in the synergy sequence DRVPPSRN>DAVPPSAN) were generated and purified as previously reported [37 , 38 (link)]. The amount of adsorbed protein was calculated via depletion assay using the bicinchoninic acid working reagent (Thermo Fisher Scientific, Waltham, MA) as previously reported [36 (link)].