Separation and purification method of scorpion venom polypeptide and use thereof
A technology for separation and purification of scorpion venom, applied in the field of separation and purification of scorpion venom polypeptides, can solve the problems of unreported, no product production and marketing, harsh conditions, etc., and achieve the effect of simple, feasible and easy control
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Embodiment 1
[0073] Determination experiment of sample pretreatment method:
[0074] Sample pretreatment method 1: Accurately weigh 100mg of scorpion venom freeze-dried powder, dissolve in 5ml of buffer solution, refrigerate overnight, centrifuge at 4°C (15000g, 20min), filter the supernatant through a 0.22μm microporous membrane, and set aside.
[0075] Sample pretreatment method 2: Accurately weigh 100mg of scorpion venom freeze-dried powder, dissolve in 5ml of buffer solution, centrifuge at 4°C (15000g, 20min), filter the supernatant through a 0.22μm microporous membrane, and set aside.
[0076] The samples of method 1 and method 2 were separated and purified in the AKTA Purifier UPC 100 protein purification system, and the results were as follows figure 1 and figure 2 shown, from figure 2 It can be seen that after the scorpion venom is dissolved in buffer, three components with relatively large peak areas can be obtained when the elution volume is 10, 15 and 18ml, and two component...
Embodiment 2
[0079] Determination experiment of sample separation and purification eluent flow rate:
[0080] After the sample was pre-treated according to method 2, the filtrate was prepared to a concentration of 10 mg / ml, and added to the chromatography column of SUPERDEX 75 prepacked column, and the Tris-HCl buffer solution with a concentration of 0.02M and a pH value of 6.8 was added to the Elution was performed at 0.5ml / min and 50.0ml / min, and the protein peak pattern was analyzed with UNICORN 5.20 software.
[0081] From image 3 and Figure 4 It can be seen that the protein peak shapes obtained at the two flow rates of 0.5ml / min and 50.0ml / min are basically the same, and the separated components are also the same. Therefore, flow rate has little effect on protein separation.
Embodiment 3
[0083] Determination experiment of sample separation and purification eluent:
[0084] After sample pretreatment according to method 2, the filtrate was prepared to a sample concentration of 10mg / ml, added to the chromatography column of SUPERDEX 75 prepacked column, and water, Tris-HCl buffer solution with a concentration of 0.02M and a pH value of 6.8 and PBS buffer with a concentration of 0.05M and a pH value of 7.2 at an elution flow rate of 1ml / min, and calculate the peak area of each protein with UNICORN 5.20 software. The water in this experiment is used as a blank control experiment. The blank control, that is, the negative control, can clearly compare and set off the changes and results of the experiment, and enhance the persuasion.
[0085] The result is as Figure 5 , Figure 6 and Figure 7 As shown, using water as the eluent, there are 2 components in about 10ml and 18ml, but the peak areas are both small. After the elution volume is 20ml, there are still ma...
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