基本描述

产品名称

Bacterial Protein Extraction Kit

别名

细菌蛋白提取试剂盒

规格或纯度

100 preps

产品介绍

细菌蛋白抽提试剂使用温和的非离子型去污剂，适用于大肠杆菌及昆虫细胞表达的重组蛋白提取。提取过程中不需进行超声破碎，有效避免了外源蛋白的污染。本产品可应用于从细菌裂解液中提取可溶性蛋白。细菌蛋白抽提试剂盒在抽提试剂的基础上添加了溶菌酶、DNaseⅠ和蛋白酶抑制剂混合物，可提高蛋白提取效率并减轻因DNA引起的粘稠现象，有效避免蛋白降解。所提取的蛋白保持了生物学活性，可进行IP、Western blot、蛋白纯化等下游操作.

B665764	Component	100T	Storage
B665764A	Bacterial Protein Extraction Reagent	100ml	RT
B665764B	Protease Inhibitor Cocktail (100x)	1ml	-20℃
B665764C	Lysozyme(50 mg/ml)	200μl	-20℃
B665764D	DNaseⅠ(1000 U/ml)	100μl	-20℃

注意事项：

1．本产品适用于从新鲜或冻存细菌和昆虫细胞中提取蛋白。

2．本品采用Tris缓冲系统，蛋白提取后的纯化操作，请使用相同的缓冲系统。

3．使用本产品获得的蛋白裂解液，可用BCA或Bradford法进行蛋白定量。

4．对于特殊的菌株，如果抽提效果不理想，可在抽提蛋白前冰冻样品。

5．根据具体情况，可在本产品中加入蛋白酶抑制剂、盐、螯合剂、还原剂等。

操作步骤：

● 昆虫细胞蛋白提取

1．低速离心收集细胞。每1 ml Bacterial Protein Extraction Reagent中加入10 μl Protease Inhibitor Cocktail即为1×工作液。

2．称量细胞湿重，按10 ml/g的量加入1×工作液。

3．重悬后，冰上孵育20分钟（冰上放置时间应根据细胞类型不同进行调整）。

4．15,000×g离心15分钟，分离可溶性蛋白。

● 细菌可溶性蛋白提取

1．5,000×g离心10分钟，收集菌体。

2．可选步骤：每1 ml Bacterial Protein Extraction Reagent中加入1 μl DNase I（1,000 U/ml）、 2 μl Lysozyme（50 mg/ml）和10 μl Protease Inhibitor Cocktail，涡旋震荡混匀。 3．按照每克菌体沉淀加入20 ml Bacterial Protein Extraction Reagent的比例，向菌体沉淀中加入抽提液，充分涡旋或用移液器上下吹打直至菌体完全重悬。

4．重悬后，室温孵育10-15分钟（放置时间应根据细胞类型不同进行调整）。

5．15,000×g离心5分钟。

6．转移上清至新的离心管中（上清中即为可溶性蛋白），进行蛋白定量及下游实验。

注意：如目的蛋白以包涵体形式存在，可使用包涵体蛋白溶解液进行溶解或优化表达条件增加可溶性蛋白的表达。

常见问题：

问题	可能原因	解决方法
目的蛋白不溶	目的蛋白表达为包涵体	优化表达条件或使用包涵体蛋白溶解液在蛋白抽提试剂中加入 Lysozyme 和 DNase I
加入 Lysozyme 后目的蛋白仍没有提取出来	温度过低	将使用试剂恢复至室温
加入 Lysozyme 后目的蛋白仍没有提取出来	Lysozyme 活性降低或失活	加入更多的 Lysozyme 或更换新的酶
提取物粘度高	DNase I 活性降低或失活	加入更多的 DNase I 或更换新的 DNase I 将镁离子的终浓度增加至 2 mM
蛋白抽提后仍有大部分蛋白存在于沉淀中	蛋白量过大	加入 Lysozyme 及 DNase I
蛋白抽提试剂有沉底析出	温度过低	将蛋白抽提试剂恢复至室温

Bacterial protein extraction reagents use mild non-ionic detergents and are suitable for extracting recombinant proteins expressed in Escherichia coli and insect cells. During the extraction process, there is no need for ultrasonic fragmentation, effectively avoiding contamination of exogenous proteins. This product can be applied to extract soluble proteins from bacterial lysates. The bacterial protein extraction kit adds a mixture of lysozyme, DNase I, and protease inhibitors to the extraction reagent, which can improve the efficiency of protein extraction and reduce the viscosity caused by DNA, effectively avoiding protein degradation. The extracted protein maintains biological activity and can be subjected to downstream operations such as IP, Western blot, and protein purification.

Component	B665764 100 preps
Bacterial Protein Extraction Reagent	100 ml
Protease Inhibitor Cocktail (100x)	1 ml
Lysozyme （50 mg/ml）	200μl
DNaseⅠ（1,000 U/ml）	100μl

Notes:
1. This product is suitable for extracting proteins from fresh or frozen bacterial and insect cells.
2. This product uses Tris buffer system. Please use the same buffer system for protein purification after extraction.
3. The protein lysis solution obtained from this product can be used for protein quantification using BCA or Bradford method.
4. For special strains, if the extraction effect is not ideal, the sample can be frozen before protein extraction.
5. Depending on the specific situation, protease inhibitors, salts, chelating agents, reducing agents, etc. can be added to this product.

Operation steps:
● Insect cell protein extraction
1. Collect cells by low-speed centrifugation. Add 10 to every 1 ml of Bacterial Protein Extraction Agent μ The Protein Inhibitor Cocktail is 1 x working fluid.
2. Weigh the wet weight of the cells and add 1 x working solution at a rate of 10 ml/g.
3. After resuspension, incubate on ice for 20 minutes (the ice storage time should be adjusted according to different cell types).
Centrifuge at 4.15000 × g for 15 minutes to isolate soluble proteins.
● Extraction of soluble bacterial proteins
1. Centrifuge for 10 minutes at a rate of 5000 × g and collect the bacterial cells.
2. Optional steps: Add 1 ml of Bacterial Protein Extraction Reagent every 1 ml μ DNase I (1000 U/ml), 2 μ Lysozyme (50 mg/ml) and 10 μ Protein Inhibitor Cocktail, vortex oscillation and mixing.

3. Add 20 ml of Bacterial Protein Extraction Reagent to each gram of bacterial precipitate, and add the extraction solution to the bacterial precipitate. Vortex thoroughly or use a pipette to blow up and down until the bacterial precipitate is completely resuspended.
4. After resuspension, incubate at room temperature for 10-15 minutes (the storage time should be adjusted according to different cell types).
5. Centrifuge at 15000 × g for 5 minutes.
6. Transfer the supernatant to a new centrifuge tube (the supernatant is soluble protein) for protein quantification and downstream experiments.
Note: If the target protein exists in the form of inclusion bodies, inclusion body protein solution can be used for dissolution or expression conditions can be optimized to increase the expression of soluble proteins.

Frequently asked questions:

Problem	Possible reasons	Resolvent
The target protein is insoluble	The target protein is expressed as an inclusion body	Optimize expression conditions or add Lysozyme and DNase I to protein extraction reagents using inclusion body protein solution
After adding Lysozyme, the target protein has not been extracted yet	Temperature too low	Restore the reagent to room temperature
After adding Lysozyme, the target protein has not been extracted yet	Lysozyme Decreased or inactivated activity	Add more Lysozymes or replace with new enzymes
Extract has high viscosity	DNase I Decreased or inactivated activity	Add more DNase I or replace with a new DNase I to increase the final concentration of magnesium ions to 2 mM
After protein extraction, most of the proteins still exist in the precipitate	Excessive protein content	Add Lysozyme and DNase I
The protein extraction reagent has sediment precipitation	Temperature too low	Restore the protein extraction reagent to room temperature

储存与运输

浓度	100 preps
储存温度	-20°C储存
运输条件	超低温冰袋运输

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批号(Lot Number)	证书类型	货号
ZJ24F0809773	分析证书	B665764

技术文档和文章

b665764_bacterial protein extraction kit product manual.pdf

b665764_细菌蛋白抽提试剂盒产品说明书.pdf

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