Biochemical Techniques and Instrumentation

Procedure

Extraction of trypsin like enzyme:

1. Take the pyloric ceaca or viscera (50 g) from the fish
2. Homogenize with cold 50mM Tris buffer (pH 8.0), containing 10mM CaCl₂ for 30 sec at 1:5 ratio.
3. Centrifuge at 10,000 rpm for 20 min at 4^oC and collect the supernatant
4. The supernatant is the “CRUDE EXTRACT”
5. Precipitate the crude extract first with 30 % ammonium sulphate. For precipitation, keep the solution overnight at refrigerator at 4^oC.
6. Then, centrifuge at 10,000 rpm for 20 min at 4 ^oC and collect the residue.
7. The residue is the “PARTIALLY PURIFIED ENZYME” of 30% ammonium sulfate fractionation.
8. Repeat the fractionation with add 40% - 70% ammonium sulfate and collect the residue.
9. Dialyze the residue dialysis membrane -110 (Hi-Media) against the 50mM Tris buffer (pH 7.5) by keeping in a refrigerator at 4 ^oC overnight.
10. Collect the dialyzed fractions, filter using syringe filter (Whatman, 0.2 µ) and store in freezer for purification by gel filtration technique.