In this method the enzyme extracts (intracellular fraction) are first subjected to a protein separation (electrophoresis in non-denaturing conditions [without SDS]), and then lipases and esterases are revealed using a- and p-naphthyl derivates of fatty acids. Released naphthol is coupled with diazotized salts (Fast Red TR salt or Fast Garnet GBC salt) to give a colored product. The PAGE procedure considered here is based on the technique described by Laemmli (5).
3.3.1. Preparation of Separation Gel (12% Acrylamide)
1. Place a comb into the assembled gel sandwich. With a pen, place a mark on the glass plate 2 cm below the teeth of the comb. This will be the level to which the separating gel is poured.
2. To a clean flask, add 3.45 mL of dH2O, 2.5 mL separation gel buffer, 4.0 mL acrylamide solution, 50 ^L of 10% APS, and 10 ^L TEMED (see Notes 4 and 5).
3. Mix thoroughly by stirring. Pour the solution smoothly to the mark and avoid the inclusion of air bubbles.
4. Immediately overlay the solution with 1.5 mL water-satured isobutanol to obtain a flat surface.
5. Allow the gel to polymerize for 30 min to 1 h at 25°C. The acrylamide is gelled when a clear line can be observed 1-2 mm below the water-satured isobutanol.
6. Rinse off the overlaying solution four times with dH2O.
3.3.2. Preparation of Stacking Gel (4°% Acrylamide)
1. To a clean flask, add 6.2 mL of dH2O, 2.5 mL stacking gel buffer, 1.33 mL acrylamide solution, 50 ^L 10% APS, and 10 ^L TEMED. Swirl the solution gently but thoroughly.
2. Rinse the top of the gel with approx 1 mL of stacking gel solution, pour off, and fill the cassettes immediately with stacking gel solution.
3. Insert the comb into cassettes; avoid trapping air bubbles.
4. Allow the gel to polymerize at least 1 h at 20°C
5. The gel can be used after 12 h or stored for 24 h at 4°C in a closed plastic bag to avoid evaporation.
1. After polymerization, fill the upper and lower reservoirs with reservoir buffer. Remove any air bubbles from the bottom of the gel so that good electric contact is achieved.
2. Insert the samples through the reservoir buffer and into the wells with a Hamilton syringe. The volume depends on the concentration of proteins in the cellular fraction. Apply approx 10 ^g of protein from each fraction. Adjust the volume to 15 ^L with phosphate buffer and mix with 15 ^L sample buffer. Load 20 ^L of these samples in each well (see Note 6).
3.3.4. Running the Gel
1. Attach the electrical leads to a suitable power supply with the proper polarity. Be sure to connect the positive lead to the lower chamber.
2. Run the gel at constant current 60 mA per gel until the tracking dye reaches 1 cm of the botton of the gel (see Note 7).
3. Turn off the power supply and disassemble the glass plates.
3.3.5. Detection of Esterase or Lipase Activity
1. Immerse the gel in a container and wash it twice with 10 mL phosphate buffer 0.1 M, pH 7.0, cooled at 10°C for 10 min.
2. Incubate the gel in 10 mL solution to detect esterase activity at 37°C for 8 h with shaking gentle
3. Wash the gel with dH2O to eliminate the complex color precipitated (see Note 8).
4. Activities were identified as colored bands on the gels (see Notes 9 and 10).
Gels may be stored between cellophane sheets or dried onto mylar sheets using a gel dryer (Bio-Rad).
3.3.7. Interpretation of Bacterial Esterase Activity Profiles
Visual comparison is the most frequently used method for interpretation of bacterial esterase electrophoretic patterns. The MF value (distance moved by the esterase band as a percentage of the distance moved by the dye front) is used for comparison only. Electrophoretic mobility variants are numbered for each esterase in decreasing order of migration (see Note 11).
Was this article helpful?