Biochemical Techniques and Instrumentation

Procedure

Extraction of sample

1. Grind 10g of fish sample in a pestle and mortar with 10-fold volume of 70% ethanol.
2. Shake the contents at 55^oC for 30min.
3. Centrifuge the contents at 10,000rpm for 10min.
4. Collect the supernatant.
5. Repeat the extraction of the pellet at 55^oC at least twice.
6. Pool the supernatants and shake vigorously.
7. Evaporate the alcohol fraction to dryness under vacuum using either a water-pump or rotary evaporator at 40-45^oC.
8. Dissolve the residue in a known volume of absolute ethanol or water for analysis.

Chromatography

1. Take a chromatography sheet carefully to a convenient size (40 x 24cm).
2. Draw a line with pencil across the sheet about 5cm away from one end.
3. Mark a number of points at intervals of 3cm.
4. Apply a small volume (25µL) of each amino acid as a separate small spot using a microsyringe.
5. A stream of hot air from a hair-dryer facilitates fast drying of spot.
6. The spot should be as small as possible for better resolution.
7. Similarly spot different known aliquots (25µL) of sample extract.
8. After spotting, place the sheet in a stainless steel trough in the chromatography chamber; firmly hold it by placing a long steel rod over the sheet. The spot-end of the sheet should be in the trough (descending chromatography).
9. Otherwise, the sheet may be rolled as a cylinder, tied together with fine thread and placed upright with the spots at the bottom in large petridish for upward movement of solvent (ascending chromatography).
10. Add the mobile organic (phase) solvent to the trough/petridish and close the chamber airtight.
11. Develop the chromatogram, preferably overnight or longer, until the solvent moves almost to the other end.
12. Note the solvent front and dry the chromatogram free of solvent in a fume chamber.
13. Spray the chromatogram with the ninhydrin reagent using an atomizer or sprayer.
14. Dry the paper for about 5min at room temperature followed by at 100^oC in an oven for 2 – 3min.
15. Amino acids appear as purple spots; while the hydroxyproline and proline amino acids give yellow coloured spots.