Method for preparing phenoloxidase active protein
A technology of active protein and phenol oxidase, applied in biochemical equipment and methods, oxidoreductase, enzyme, etc., can solve the problems of complex and expensive equipment, achieve the effect of improving product purity, simplifying production process, and low cost
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Embodiment 1
[0032] Embodiment 1 adopts the following steps to prepare:
[0033] (1) Take 40ml of Limulus plasma and place it in a water bath at 10°C, add dropwise 57.7ml of 100% saturated ammonium sulfate solution, stir while adding, after the dropwise addition, put it in a refrigerator at 4°C for 1 hour;
[0034] (2) After standing for 1 hour, take it out, and use a high-speed refrigerated centrifuge to centrifuge for 30 minutes at 4°C and 11,000r / min to obtain Limulus plasma hemocyanin, which is stored at 4°C for later use;
[0035] (3) Take Limulus plasma hemocyanin and dissolve it in 0.01mol / L, pH=7 PBS buffer (protein concentration is 10mg / mL), put it into a MW3500Da dialysis bag, dialyze overnight, collect the solution, and store it at 4°C. spare;
[0036] (4) Purify the solution collected in step (3) through a SephacrylS-100 propylene dextran gel column, elute with 50mmol / L Tris-HCl at pH=7.0, collect the solution that absorbs at 280nm, and obtain blood Blue protein solution is p...
Embodiment 2
[0038] Embodiment 2 adopts the following steps to prepare:
[0039] (1) Take 40ml of Limulus plasma and place it in a water bath at 10°C, add dropwise 57.7ml of 100% saturated ammonium sulfate solution, stir while adding, after the dropwise addition, put it in a refrigerator at 4°C for 1 hour;
[0040] (2) After standing for 1 hour, take it out, and use a high-speed refrigerated centrifuge to centrifuge for 30 minutes at 4°C and 11,000r / min to obtain Limulus plasma hemocyanin, which is stored at 4°C for later use;
[0041] (3) Take Limulus plasma hemocyanin and dissolve it in 0.01mol / L, pH=7 PBS buffer (protein concentration is 10 mg / mL), put it into a MW3500Da dialysis bag, dialyze overnight, collect the solution, and store it at 4°C. spare;
[0042] (4) Purify the solution collected in step (3) through a SephacrylS-100 propylene dextran gel column, elute with 50mmol / L Tris-HCl at pH=7.0, collect the solution that absorbs at 280nm, and obtain blood Blue protein solution is ...
Embodiment 3
[0044] Embodiment 3 adopts the following steps to prepare:
[0045] (1) Take 40ml of Limulus plasma and place it in a water bath at 10°C, add dropwise 57.7ml of 100% saturated ammonium sulfate solution, stir while adding, after the dropwise addition, put it in a refrigerator at 4°C for 1 hour;
[0046] (2) After standing for 1 hour, take it out, and use a high-speed refrigerated centrifuge to centrifuge for 30 minutes at 4°C and 11,000r / min to obtain Limulus plasma hemocyanin, which is stored at 4°C for later use;
[0047] (3) Take Limulus plasma hemocyanin and dissolve it in 0.01mol / L, pH=7 PBS buffer (protein concentration is 10 mg / mL), put it into a MW3500Da dialysis bag, dialyze overnight, collect the solution, and store it at 4°C. spare;
[0048] (4) Purify the solution collected in step (3) through a SephacrylS-100 propylene dextran gel column, elute with 50mmol / L Tris-HCl at pH=7.0, collect the solution that absorbs at 280nm, and obtain blood Blue protein solution is ...
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