Cholesterol Homeostasis by Ingrid C. Gelissen & Andrew J. Brown

Author:Ingrid C. Gelissen & Andrew J. Brown
Language: eng
Format: epub
Publisher: Springer New York, New York, NY

2.BCA Protein Assay Kit. Store at room temperature.

2.3 In Vitro Reconstitution of Lysosome–Peroxisome Membrane Contact (LPMC) and 3H-Cholesterol Transfer

1.Ni Sepharose beads. Store at 4 °C.

2.Wash buffer: 20 mM HEPES (pH 7.2), 2 mM EGTA (pH 8.0), 250 mM sucrose, 1 mM MgCl2, 50 mM KCl, 1 mM DTT (see Note 8 ). Start with preparing 100 mL of 0.5 M EGTA (pH 8.0). Weigh 19.018 g of EGTA and transfer to a 100-mL beaker containing about 70 mL of ultrapure water. Stir vigorously on a magnetic stirrer while adjusting pH with NaOH pellets. The suspension will become clear when the pH is approximately 8.0. Make up to 100 mL with ultrapure water and sterilize by autoclaving. Store EGTA solution at room temperature.

For the wash buffer, weigh 8.55 g of sucrose , 0.02 g of MgCl2⋅6H2O, 0.37 g of KCl, and 0.031 g of DTT and transfer to a 100-mL beaker containing about 80 mL of ultrapure water. Stir vigorously on a magnetic stirrer until everything dissolves completely. Then add 2 mL of 1 M HEPES (pH 7.2; see above under 14) and 0.4 mL of 0.5 M EGTA (pH 8.0; see above). Make up to 100 mL with ultrapure water. Store at 4 °C.

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