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[交流] 【求助/交流】蛋白纯化已有2人参与

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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1楼 2010-04-16 12:55:15

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scelab(金币+2):热心虫友，欢迎多来交流~~~:tiger06::tiger28::tiger23: 2010-04-16 22:00
arniekyo(金币+1): 2010-08-15 14:56:38

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Originally posted by arniekyo at 2010-04-16 12:55:15:
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

如果是买的柱子说明书上会有告诉你这些缓冲液的配方
如果没有就参照分子克隆上的配方

[ Last edited by 小白3521 on 2010-4-16 at 14:52 ]

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2楼2010-04-16 14:50:31

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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
arniekyo(金币+1): 2010-04-17 19:42
arniekyo(金币+1): 2010-04-18 14:57
arniekyo(金币+1): 2010-06-18 09:34:05

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.

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3楼2010-04-16 15:57:03

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