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I am trying to treat mouse pancreatic islets with palmitate these days. I dissolve Palmitic acid (not palmitate sodium) in 100% ethanol, vortex it, heat at 60C for a few mins and vortex again to dissolve it completely. I then couple it to 1% BSA (fatty acid free-from Roche: Cat# 10775835001) such that the final Palmitate concentration is 0. 5 mM. BSA solution is prepared in complete CMRL medium containing 10% FCS.
5. Prepare reaction mixture from stock solutions for the number of reactions (400 L/reaction +10%) at room temperature with the following final concentration: 100 mM sucrose, 10 mM Tris-HCl, 5 mM KH2PO4, 0. 2 mM EDTA, 5 mM NAM, 1 M TSA, 0. 3% fatty acid-free BSA, 80 mM KCl, 1 mM MgCl2, 2 mM L-carnitine, 0. 1 mM malate, 0. 05 mM coenzyme A, and adjust to pH 8. 0 (Table 1). This is the entire reaction mixture without DTT, ATP and substrate, which are added immediately before use.
Gemini’s fatty acid-free BSA is a highly purified, lyophilized powder. This product is processed to provide a minimum of 98% albumin and to be essentially free of non-esterified fatty acids. The pH is adjusted to 7. 0. It is used as a blocking reagent in EIA, RIA and ELISA systems, where serum fatty acids may interfere with the application.
All tissue culture media were from Life Technologies, Gibco BRL. Bovine pituary extract was purchased from Clonetics. Fatty-acid-free bovine serum albumin (BSA), 3,3,5-triiodo-L-thyronine, tocopherol phosphate disodium, the lipid TLC and high performance liquid chromatography (HPLC) standards, and all the fatty acids used were purchased from Sigma. The radioactive fatty acids were purchased from American Radiolabeled Chemicals, St.