HPLC grade solvents were purchased from Sigma-Aldrich unless otherwise noted. Sequencing grade trypsin was purchased from Promega (Fitchburg, WI). Prodan (6-Propionyl-2 – dimethylaminonaphthalene) was a product of Anaspec Inc (Fremont, CA, catalog #88212, lot #64774). Fatty acid-free human serum albumin (catalog # A3782) and 40% methylglyoxal solution were obtained from Sigma-Aldrich. Lipidex-1000 was acquired from PerkinElmer (Waltham, MA).
Lipidex 1000
Manufactured by PerkinElmer
Lipidex-1000 is a liquid chromatography column used for the separation and purification of lipids. It is designed to provide efficient and reliable lipid separation for analytical and preparative applications.
Automatically generated - may contain errors
3 protocols using lipidex 1000
1
Preparation of Prodan-Conjugated Albumin
2
Acyl-CoA Binding Assay for Atg37 and Atg30
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Recombinant His-Atg37ΔC (WT and acbs mutant) and GST-Atg30 were purified, as described above. Then they were mixed at a final concentration of 0.07 µM with increasing concentrations of [14C]palmitoyl-CoA (NEC555010UC; PerkinElmer) or [14C]oleoyl-CoA (NEC651A010UC; PerkinElmer) ranging from 1.5 µM to 9.0 µM in 100 µl of binding buffer (10 mM potassium phosphate, pH 7.4). The reaction mixture was incubated for 30 min at 30°C. Tubes were kept on ice for 10 min and mixed with 600 µl of ice-cold 50% slurry of lipidex-1000 (6008301; PerkinElmer) in binding buffer to bind unbound acyl-CoA. The samples were incubated on ice for 10 min and centrifuged at 12,000 g for 5 min at 4°C, and 200 µl of supernatant (bound acyl-CoA) was taken for analysis of radioactivity counts. Assays were performed in triplicate with protein samples and blanks at each concentration of acyl-CoA. Radioactivity counts of blanks were subtracted from the counts of samples.
3
Delipidation of PPARγ Ligand Binding Domain
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To delipidate PPARγ LBD, purified protein was diluted to 0.8 mg ml−1 and batched with Lipidex 1000 (Perkin-Elmer) at an equal volume. This mixture was batched for 1 h at 37 °C and 100 rpm. Immediately following this treatment, protein was pulled through a gravity column by syringe. To increase yield, it was found that the speed of elution was important; protein could not remain on the resin at room temperature in excess of 3 min. Two more column volumes of pre-warmed 25 mM MOPS, 25 mM KCl, and 1 mM EDTA were also pulled through in the same manner. Quality of delipidation was then estimated by 19F NMR, and loss of lipid can be most easily detected by a reduction in the peak at −84.1 ppm. When non-delipidated protein is used in this report it is labeled as bound to E. coli lipids.
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