Sehadex G-50 Lab Report
Gel filtration chromatography on Sephadex G-50
The crude broth obtained after fermentation was subjected to ammonium sulphate precipitation at 70% (w/v). The pellet so obtained was resuspended in cold saline (2 ml) and dialysed. The dialysed enzyme was loaded onto a column of Sephadex G-50 (120 cm × 1.0 cm) equilibrated with 10 mM Tris-HCl buffer, pH 8. The column was eluted at a flow rate of 1 ml / 6 min. The elution profile of gel filtration chromatography is shown in the (Fig: 1). The fractions collected were determined for its total protein concentration and fibrinolytic enzyme activity. The active fraction obtained was pooled together, concentrated by lyophilization and used as purified fibrinolytic enzyme for subsequent studies. The summary of purification steps involved for fibrinolytic protease is presented in the Table: 1.
Anion exchange chromatography on DEAE Sephadex A-50
The dialyzed enzyme was chromatographed on a column of DEAE Sephadex A-50. The sample was loaded onto a column of DEAE Sephadex A-50 (24 cm × 2.0 cm) equilibrated with 20 mM Tris-HCl buffer, pH 8. The absorbed protein solution was eluted …show more content…
Several bands were observed in the case of ammonium sulphate precipitate (Fig: 3), while purified protease showed a single band on SDS-PAGE, indicating a homogeneous preparation. The molecular weight of the fibrinolytic protease was determined by comparison of the migration distances of standard marker proteins (Lysozyme 14.4 kDa, Soybean Trypsin Inhibitor 21.5 kDa, Carbonic anhydrase 31.0 kDa, Ovalbumin 45.0 kDa, Bovine Serum Albumin 66.2 kDa and Phosphorylase b 97.4 kDa). Depending on the relative mobility, the molecular weight of the protein band was calculated to be around 31 kDa. Thus, it was concluded that our fibrinolytic protease enzyme has a molecular weight of 31
The crude broth obtained after fermentation was subjected to ammonium sulphate precipitation at 70% (w/v). The pellet so obtained was resuspended in cold saline (2 ml) and dialysed. The dialysed enzyme was loaded onto a column of Sephadex G-50 (120 cm × 1.0 cm) equilibrated with 10 mM Tris-HCl buffer, pH 8. The column was eluted at a flow rate of 1 ml / 6 min. The elution profile of gel filtration chromatography is shown in the (Fig: 1). The fractions collected were determined for its total protein concentration and fibrinolytic enzyme activity. The active fraction obtained was pooled together, concentrated by lyophilization and used as purified fibrinolytic enzyme for subsequent studies. The summary of purification steps involved for fibrinolytic protease is presented in the Table: 1.
Anion exchange chromatography on DEAE Sephadex A-50
The dialyzed enzyme was chromatographed on a column of DEAE Sephadex A-50. The sample was loaded onto a column of DEAE Sephadex A-50 (24 cm × 2.0 cm) equilibrated with 20 mM Tris-HCl buffer, pH 8. The absorbed protein solution was eluted …show more content…
Several bands were observed in the case of ammonium sulphate precipitate (Fig: 3), while purified protease showed a single band on SDS-PAGE, indicating a homogeneous preparation. The molecular weight of the fibrinolytic protease was determined by comparison of the migration distances of standard marker proteins (Lysozyme 14.4 kDa, Soybean Trypsin Inhibitor 21.5 kDa, Carbonic anhydrase 31.0 kDa, Ovalbumin 45.0 kDa, Bovine Serum Albumin 66.2 kDa and Phosphorylase b 97.4 kDa). Depending on the relative mobility, the molecular weight of the protein band was calculated to be around 31 kDa. Thus, it was concluded that our fibrinolytic protease enzyme has a molecular weight of 31