Bovine albumin

Bovine serum albumin (BSA) and palmitate conjugated to BSA were prepared according to a previously described protocol (52 (link), 54 (link), 55 (link)). Briefly, to prepare the BSA solution, 20% (w/v) fatty acid free bovine albumin (MP Biomedicals, Cat #0219477205) was gradually added to Ultra Pure water (Biological Industries, Cat #01-866-1A) at 52°C with gentle agitation until the BSA was fully dissolved, yielding a transparent yellow solution. To prepare a palmitate-BSA solution, 2.6% (w/v) of palmitic acid (Sigma-Aldrich, Cat #P0500) was added to 0.1 M NaOH (Sigma-Aldrich, Cat #221465) and heated to 70°C until fully dissolved, yielding a concentration of 100 mM. To conjugate palmitate to BSA, the 100 mM stock of palmitate in 0.1 M NaOH was added to 20% BSA at a ratio of 1:9 and heated at 55°C for 10 min. After conjugation, both the 20% (w/v) BSA solution and 10 mM Palmitate-BSA solutions were filter sterilized using a 0.45 μm PES filter (Celltreat, Cat #229709). Solutions were then aliquoted and stored at −20°C. Before use, solutions were warmed to 37°C. The total amount of BSA was held constant in all conditions by mixing ratios of fatty acid free BSA to palmitate-BSA as follows: BSA only (4:0), 0.1 mM palmitate-BSA (3:1), 0.2 mM palmitate-BSA (2:2), and 0.4 mM palmitate-BSA (0:4).