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【求助/交流】蛋白纯化 已有2人参与
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| 我现在要提取原核表达的蛋白，不知道相关的试剂上样buffer，漂洗buffer，洗脱buffer怎么样配制，是His标签的包涵体，请教高手指点 |
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小白3521
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2楼2010-04-16 14:50:31
jingcp200320
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scelab(金币+3):热心虫友，欢迎多来交流~~~:tiger06::tiger28::tiger23: 2010-04-16 22:00
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Recipes Buffer Stock Solutions (10X) To prepare the buffer solutions described below, you need to prepare sodium phosphate stock solutions: Stock Solution A (10X) 200 mM sodium phosphate, monobasic (NaH2PO4) 5 M NaCl Dissolve 27.6 g sodium phosphate, monobasic (NaH2PO4) and 292.9 g NaCl in 900 ml of deionized water. Mix well and adjust the volume to 1 L with deionized water. Store solution at room temperature. Stock Solution B (10X) 200 mM sodium phosphate, dibasic (Na2HPO4) 5 M NaCl Dissolve 28.4 g sodium phosphate, dibasic (Na2HPO4) and 292.9 g of NaCl in 900 ml of deionized water. Mix well and adjust the volume to 1 L with deionized water. Store solution at room temperature. 5X Native Purification Buffer 250 mM NaH2PO4, pH 8.0 2.5 M NaCl Prepare 200 ml solution as follows: 1. To 180 ml deionized water, add Sodium phosphate, monobasic 7 g NaCl 29.2 g 2. Mix well and adjust the pH with NaOH to pH 8.0. 3. Bring the final volume to 200 ml with water. 4. Store buffer at room temperature. 3 M Imidazole pH 6.0 3 M Imidazole 500 mM NaCl 20 mM Sodium Phosphate Buffer, pH 6.0 Prepare 100 ml solution as follows: 2. To 90 ml deionized water, add Imidazole 20.6 g Stock Solution A (10X) 8.77 ml Stock Solution B (10X) 1.23 ml 3. Mix well and adjust the pH to 6.0 with HCl or NaOH as necessary. 4. Bring the final volume to 100 ml with water. If the solution forms a precipitate, heat solution until the precipitate dissolves. 5. Store buffer at room temperature. Continued on next page 18 Recipes, Continued Guanidinium Lysis Buffer 6 M Guanidine Hydrochloride 20 mM Sodium Phosphate, pH 7.8 500 mM NaCl Prepare 100 ml solution as follows: 1. To 90 ml deionized water, add Stock Solution A (10X) 0.58 ml Stock Solution B (10X) 9.42 ml Guanidine Hydrochloride 57.3 g 2. Stir the solution until completely dissolved. Adjust the pH to 7.8 using 1 N NaOH or 1 N HCl. 3. Bring the volume to 100 ml and filter sterilize the buffer using a 0.45 μm filter (autoclaving the solution will alter the pH of the buffer). 4. Store buffer at room temperature. Denaturing Binding Buffer 8 M Urea 20 mM Sodium Phosphate pH 7.8 500 mM NaCl Prepare 100 ml solution as follows: 1. To 90 ml deionized water, add Stock Solution A (10X) 0.58 ml Stock Solution B (10X) 9.42 ml Urea 48.1g 2. Stir the solution with gentle heating (50-60°C, do not overheat) until completely dissolved. When cooled to room temperature, adjust the pH to 7.8 using 1 N NaOH or 1 N HCl. 3. Bring the volume to 100 ml and filter sterilize the buffer using a 0.45 μm filter (autoclaving the solution will alter the pH of the buffer). 4. Store buffer at room temperature. Continued on next page 19 Recipes, Continued Denaturing Wash Buffer 8 M Urea 20 mM Sodium Phosphate, pH 6.0 500 mM NaCl Prepare 100 ml solution as follows: 1. To 90 ml deionized water, add Stock Solution A (10X) 7.38 ml Stock Solution B (10X) 2.62 ml Urea 48.1g 2. Stir the solution with gentle heating (50-60°C, do not overheat) until completely dissolved. Adjust the pH to 6.0 using 1 N NaOH or 1 N HCl. 3. Bring the volume to 100 ml and filter sterilize the buffer using a 0.45 μm filter (autoclaving the solution will alter the pH of the buffer). 4. Store buffer at room temperature. Denaturing Elution Buffer 8 M Urea 20 mM Sodium Phosphate, pH 4.0 500 mM NaCl Prepare 100 ml as follows: 1. To 90 ml deionized water, add Stock Solution A (10X) 10 ml Urea 48.1g 2. Stir the solution with gentle heating (50-60°C, do not overheat) until completely dissolved. Adjust the pH to 4.0 using 1 N NaOH or 1 N HCl. 3. Bring the volume to 100 ml and filter sterilize the buffer using a 0.45 μm filter (autoclaving the solution will alter the pH of the buffer). 4. Store buffer at room temperature. |
3楼2010-04-16 15:57:03
jingcp200320
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4楼2010-04-16 15:57:28
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