Chromogenic Method

2.2.1. Substrates: a- and ß-Naphthyl Derivatives of Fatty Acids

1. 0.1 M Sodium phosphate buffer, pH 7.0.

a. Solution A (0.2 M NaH2PO4: Weigh 27.6 g NaH2PO4-H2O and make to 100 mL with distilled water (dH2O).

b. Solution B (0.2 MNa2HPO4): Weigh 53.05 g of Na2HPO4-7H2O and make to 100 mL with dH2O.

c. To prepare 1 L of 0.1 M phosphate buffer (pH 7.0), mix 195 mL of solution A and 305 mL of solution B and bring to 1000 mL with dH2O.

2. 66 mM a- and ß-naphthyl substrate in ethanol/water (1:1). Always use a freshly prepared solution.

3. 10% (w/v) Sodium dodecyl sulfate (SDS): dissolve 10 g SDS in 100 mL dH2O with gentle stirring. Store at room temperature. Solution may become cloudy at temperatures below 20°C, but warming to 30°C and mixing may restore clarity.

4. 10% Fast Garnet GBC solution: dissolved 5 mg of Fast Garnet GBC (Sigma) in 1 mL 10% SDS. Always use a freshly prepared solution.

2.2.2. Substrates: p-Nitrophenyl Derivatives of Fatty Acids

1. 0.15 M Sodium phosphate buffer, pH 7.0.

a. Solution A (0.2 M NaH2PO4): Weigh 27.6 g NaH2PO4-H2O and make to 100 mL with distilled water (dH2O).

b. Solution B (0.2 MNa2HPO4): Weigh 53.05 g of Na2HPO4-7H2O and make to 100 mL with dH2O.

c. To prepare 1 L of 0.15 M phosphate buffer (pH 7.0), mix 293 mL of solution A and 457 mL of solution B and bring to 1000 mL with dH2O.

2. Solvent mixture: acetone/water at a concentration of 1:1

3. Substrate solution: 22.5 mM pNP derivate of fatty acids in solvent mixture. Always use a freshly prepared solution.

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