Imagequant analysis software

Total genomic DNA was digested with SacII (New England BioLabs) for strains containing pBR322-derived plasmids or NheI (New England BioLabs) for pCB104-derived plasmids. In both cases, plasmids lack restriction sites for these enzymes. Samples were then extracted with one volume of chloroform before equal cell equivalents were electrophoresed through 1.0% agarose gel in 1× TBE (Tris-borate-EDTA, pH 8.0) at 1 V/cm. DNA in the gels was transferred to a Hybond N+ nylon membrane, and the plasmid DNA was detected by probing with either ³²P-labeled pBR322 or pCL01 plasmid DNA prepared by random-primer labeling (Agilent Technologies) using α³²P-labeled-dCTP (3000 Ci/mmol; PerkinElmer) and visualized using a STORM PhosphorImager with its associated ImageQuant analysis software (Amersham Biosciences).

Crude cellular extracts (35 µg) or organelle fraction lysates (10 µg) were incubated at 37°C in 100 µL reaction mixture containing 20 mM NAD, 10 mM CoA-SH, 1 mM FAD, 100 mM KCN, 2% Triton, Tris-HCl 100 mM pH 7.4. The reaction was started by addition of 1 µL of ¹⁴C_α-palmitoyl-CoA 10 µM (20 µCi/ml, Sigma-Aldrich). The reaction was stopped by addition of 100 µL KOH 5 M. After saponification (one hour at 65°C), and acidification (addition of 100 µL H₂SO₄ 36 N), FA were extracted with 2 mL of CHCl₃. Extracts were evaporated (under N₂), dissolved in 50 µL CHCl₃/CH₃OH 2∶1 (v/v) and the compounds were separated by thin layer chromatography (TLC) on 10×10 cm silica gel plates (Merck) in 75∶24∶1 hexane/diethyl ether/acetic acid (v/v/v). Revelation was done using Phosphorimager SI system (Amersham Biosciences). Analysis of ¹⁴C_α-palmitoyl-CoA consumption were done using ImageQuant analysis software (Amersham Biosciences).