Method for separating and purifying cathepsin L in dorsal muscle of carp
A technology for separation and purification of cathepsin, applied in the field of separation and purification of cathepsin L in carp back muscle, achieving high yield, good separation effect and good purity
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Embodiment 1
[0024] Take 50 g of the back white meat of fresh and alive carp, add 500 mL of 20 mM Tris-HCl buffer solution (pH 7.0), homogenize, and centrifuge at 6000×g for 20 min at 3°C to obtain a supernatant. The supernatant was placed in a water bath at 60° C. for 4 minutes, cooled in an ice bath, and centrifuged at 8000×g for 20 minutes at 3° C. to obtain a supernatant. Load the supernatant as a crude enzyme solution onto a Phenyl-Sepharose column (inner diameter 1.5cm, height 20cm) that has been equilibrated with the eluent, and elute with 20mM Tris-HCl buffer (pH7.5) at a flow rate of 0.5ml / min. The eluates with enzymatic activity were collected, combined, and further purified by dialysis against 20 mM Tris-HCl buffer (pH 7.5). The obtained sample was then loaded onto a pre-equilibrated DEAE-Bio-Gel A column (inner diameter 1.5 cm, height 15 cm), eluted with 20 mM Tris-HCl buffer (pH 7.5) overnight, and washed with a gradient of 0-300 mM NaCl Remove (total volume 300ml). Coll...
Embodiment 2
[0026] Take 50 g of the back white meat of fresh and alive carp, add 250 mL of 20 mM Tris-HCl buffer solution (pH 6.8), homogenize, and centrifuge at 6200×g for 15 min at 4° C. to obtain the supernatant. The supernatant was placed in a water bath at 55° C. for 5 minutes, cooled in an ice bath, and centrifuged at 8300×g for 15 minutes at 4° C. to obtain a supernatant. Load the supernatant as a crude enzyme solution onto a Phenyl-Sepharose column (inner diameter 1.5cm, height 20cm) that has been equilibrated with the eluent, and elute with 20mM Tris-HCl buffer (pH7.5) at a flow rate of 0.5ml / min. The eluates with enzymatic activity were collected, combined, and further purified by dialysis against 20 mM Tris-HCl buffer (pH 7.5). The obtained sample was then loaded onto a pre-equilibrated DEAE-Bio-Gel A column (inner diameter 1.5 cm, height 15 cm), eluted with 20 mM Tris-HCl buffer (pH 7.5) overnight, and washed with a gradient of 0-300 mM NaCl Remove (total volume 300ml). Co...
Embodiment 3
[0028]Take 45g of the back white meat of fresh and alive carp, add 360mL of 20mM Tris-HCl buffer solution (pH7.5), homogenize, and centrifuge at 6500×g for 18min at 5°C to obtain the supernatant. The supernatant was placed in a water bath at 65° C. for 3 minutes, cooled in an ice bath, and centrifuged at 8500×g for 17 minutes at 5° C. to obtain a supernatant. Load the supernatant as a crude enzyme solution onto a Phenyl-Sepharose column (inner diameter 1.5cm, height 20cm) that has been equilibrated with the eluent, and elute with 20mM Tris-HCl buffer (pH7.5) at a flow rate of 0.5ml / min. The eluates with enzyme activity were collected, combined, and further purified by dialysis with 20 mM Tris-HCl buffer (pH 7.5). The obtained sample was then loaded onto a pre-equilibrated DEAE-Bio-Gel A column (inner diameter 1.5 cm, height 15 cm), eluted with 20 mM Tris-HCl buffer (pH 7.5) overnight, and washed with a gradient of 0-300 mM NaCl Remove (total volume 300ml). Collect the elua...
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