tailieunhanh - Genetic Engineering Basics New Applications and Responsibilities Part 11

Tham khảo tài liệu 'genetic engineering basics new applications and responsibilities part 11', khoa học tự nhiên, công nghệ sinh học phục vụ nhu cầu học tập, nghiên cứu và làm việc hiệu quả | 190 Genetic Engineering - Basics New Applications and Responsibilities 11. Semipurification of recombinant hormones Figure 11 shows the process of purification or downstream that was followed for each recombinant hormone produced. Each culture medium containing the recombinant hormone was ultra-dialyzed and ultraconcentrated. The pore membrane used was 14 kDa. At the same time it was ultradyalized. The ultraconcentrate obtained was lyophilized to preserve the samples and all the powder was recovered in 50 mL plastic tubes and weighed. Total protein was quantified in each lyophilized culture medium with the Bradford method 52 . Samples were stored at -20 C until use. Each sample was prepared for loading into a chromatographic column. Five mg of the total protein from each sample was adjusted to the conditions of the loading buffer. A column was loaded with Q-Sepharose fast flow anion exchange resin column filling with resin was carried out by gravity flow. After passing the sample washing was done with 10 mL of loading buffer. To recover the proteins from the column 15 mL of elution buffer loading buffer plus NaCl was passed with an ionic strength increased sequentially with NaCl. Total protein was measured by the Bradford method 52 for each of the fractions recovered to see what percentage of the total they represented. The collected fractions were visualized on discontinuous polyacrylamide gel concentrations of 4-15 under denaturing conditions SDS-PAGE and stained with Coomassie and silver techniques 53 . The fractions were subjected to lyophilization the powder was recovered and weighted total protein was measured by the Bradford method. In addition using PAGE-SDS the percentage of purity of the monomer was determined with ImageJ software. The samples were stored at -20 C until used for further analysis or to determine their biological activity. 12. Testing the biological activity of recombinant hormones Lactogenic activity bioassay The biological activity .

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