基本描述

英文名称

Mammalian Protein Extraction Kit

储存温度

-20°C储存

运输条件

超低温冰袋运输

产品介绍

本产品能够快速、温和、高效地裂解哺乳动物细胞，有效提取细胞浆和细胞核蛋白。该试剂使用温和配方保证所提取蛋白保持生物学活性并可应用于多种蛋白分析试验，如报告基因和酶活性测定、免疫检测、蛋白纯化等。提取后蛋白可采用BCA法进行蛋白定量分析。试剂盒中带有蛋白酶抑制剂混合物，可有效避免蛋白提取过程中蛋白的降解。

M665813	Component	100 T	Storage
M665813A	Mammalian Protein Extraction Reagent	100 mL	RT
M665813B	Protease Inhibitor Cocktail (100×)	1 mL	-20℃. Avoid freeze/thaw cycle.

注意事项

1．本产品可有效裂解细胞培养板培养的贴壁细胞（无需刮取）以及离心收集的悬浮细胞，较反复冻融或超声法具有更高提取效率。但对于组织蛋白的抽提，建议使用组织蛋白抽提试剂盒（CW0891）。

2．表1中所列的是贴壁细胞蛋白提取的最佳使用量，先收集细胞可以减少试剂用量从而获得更高的蛋白浓度。

3．也可以根据细胞数量估计抽提试剂的使用量。如2×106 个Hela细胞约重20 mg，需要加入 200 μl抽提试剂。

4．通过本产品提取的蛋白，可通过BCA法进行蛋白定量分析。

操作步骤

● 贴壁细胞蛋白抽提

1．请在蛋白抽提前取出实验所需Mammalian Protein Extraction Reagent进行预冷。

2．小心倾去贴壁细胞的培养液，使用PBS漂洗细胞。

3．加入适量Mammalian Protein Extraction Reagent（抽提蛋白前2-3分钟按照1:99比例加入Protease Inhibitor Cocktail），在冰上用枪头吹打贴壁细胞，将裂解液转移至离心管中，冰上孵育20分钟，让细胞充分裂解（试剂使用量请参考附表1，冰上放置时间应根据细胞类型不同进行调整）。

4．14,000×g离心5-10分钟。

5．转移上清液至新管中，用于进一步分析。

● 悬浮细胞蛋白提取

1．请在蛋白抽提前取出实验所需Mammalian Protein Extraction Reagent进行预冷。

2．将悬浮细胞2,500×g，离心10分钟，弃去上清。使用PBS漂洗细胞。2,500×g，离心 10分钟，弃去上清。

3．加入适量Mammalian Protein Extraction Reagent，抽提蛋白前2-3分钟按照1:99比例加入Protease Inhibitor Cocktail，即1×工作液。

4．每100 mg细胞加入至少1 ml 1×工作液。如提取的样本量较大，可首先使用少量1×工作液重悬细胞，然后加入剩余工作液。

5．吹打均匀后，冰上放置20分钟，让细胞充分裂解（冰上放置时间应根据细胞类型不同进行调整）。

6．14,000×g离心15分钟。

7．转移上清至新管中，进行下一步分析。

表1. 抽提试剂使用量推荐表

细胞培养板类型或平皿类型	抽提试剂使用量
100 mm	500-1,000 µl
60 mm	250-500 µl
6 孔培养板	200-400 µl /孔
24 孔培养板	100-200 µl /孔
96 孔培养板	50-100 µl /孔

表2. 常见问题及解决办法

问题	可能原因	解决方法
低提取率	低蛋白表达量	优化转染系统
低提取率	试剂使用量不够	增加抽提试剂使用量
低提取率	试剂无法溶解细胞膜	增加裂解时间或者加大晃动幅度
无法获得膜蛋白	本产品更适合提取核浆蛋白	使用真核细胞膜蛋白抽提试剂盒

This product can rapidly, gently, and efficiently lyse mammalian cells, effectively extracting cytoplasmic and nuclear proteins. This reagent uses a mild formula to ensure that the extracted protein maintains biological activity and can be applied to various protein analysis experiments, such as reporter gene and enzyme activity determination, immune detection, protein purification, etc. The extracted protein can be quantitatively analyzed using the BCA method. The reagent kit contains a mixture of protease inhibitors, which can effectively prevent protein degradation during the protein extraction process.

M665813	Component	100 T	Storage
M665813A	Mammalian Protein Extraction Reagent	100 mL	RT
M665813B	Protease Inhibitor Cocktail (100×)	1 mL	-20℃. Avoid freeze/thaw cycle.

precautions

1. This product can effectively lyse adherent cells cultured on cell culture plates (without scraping) and suspended cells collected by centrifugation, with higher extraction efficiency than repeated freeze-thaw or ultrasound methods. But for the extraction of tissue proteins, it is recommended to use the tissue protein extraction kit (CW0891).
The optimal dosage for protein extraction from adherent cells is listed in Table 1. Collecting cells first can reduce the amount of reagents used to obtain higher protein concentrations.
3. The amount of extraction reagents used can also be estimated based on the number of cells. If 2 × 106 Hela cells weigh about 20 mg, 200 need to be added μ Extract reagents.
4. The protein extracted from this product can be quantitatively analyzed using the BCA method.

Operation steps
● Protein extraction from adherent cells
1. Please remove the required Mammalian Protein Extraction Agent for pre cooling before protein extraction.
2. Carefully pour out the culture medium of adherent cells and rinse the cells with PBS.
3. Add an appropriate amount of Mammalian Protein Extraction Reagent (add Protein Inhibitor Cocktail in a 1:99 ratio 2-3 minutes before protein extraction), blow adherent cells on ice with a gun tip, transfer the lysate to a centrifuge tube, incubate on ice for 20 minutes, and allow the cells to fully lyse (please refer to Appendix 1 for the amount of reagent used, and the time for placing on ice should be adjusted according to different cell types).
4. Centrifuge at 14000 × g for 5-10 minutes.
5. Transfer the supernatant to a new tube for further analysis.
● Suspension cell protein extraction
1. Please remove the required Mammalian Protein Extraction Agent for pre cooling before protein extraction.
2. Suspend 2500 × g of cells, centrifuge for 10 minutes, and discard the supernatant. Rinse cells with PBS. 2500 × g, centrifuge for 10 minutes, discard the supernatant.
3. Add an appropriate amount of Mammalian Protein Extraction Agent, and 2-3 minutes before protein extraction, add Protein Inhibitor Cocktail in a ratio of 1:99, which is 1 x working solution.
4. Add at least 1 ml of 1x working solution to every 100 mg of cells. If the extracted sample size is large, a small amount of 1x working solution can be used to resuspend the cells first, and then the remaining working solution can be added.
5. After blowing evenly, place it on ice for 20 minutes to allow the cells to fully lyse (the time for placing it on ice should be adjusted according to different cell types).
6. Centrifuge at 14000 × g for 15 minutes.
7. Transfer the supernatant to a new tube for further analysis.

Table 1. Recommended usage of extraction reagents

Cell culture plate type or dish type	Extraction reagent usage
100 mm	500-1,000 µl
60 mm	250-500 µl
6-well culture plate	200-400 µl /well
24-well culture plate	100-200 µl /well
96-well culture plate	50-100 µl /well

Table 2. Common Problems and Solutions

Problem	Possible reasons	Resolvent
Low extraction rate	Low protein expression level	Optimize transfection system
Low extraction rate	Insufficient reagent usage	Increase the usage of extraction reagents
Low extraction rate	Reagent unable to dissolve cell membrane	Increase cracking time or increase shaking amplitude
Unable to obtain membrane protein	This product is more suitable for extracting nuclear plasma protein	Using eukaryotic cell membrane protein extraction kit

质检证书（CoA,COO,BSE/TSE 和分析图谱)

C of A & Other Certificates(BSE/TSE, COO):

输入批号以搜索分析图谱:

技术文档和文章

m665813_mammalian protein extraction kit.pdf

溶液计算器

摩尔浓度计算器

计算溶液所需的质量、体积或浓度。

质量

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浓度

x

体积

x

分子量

g/mol

稀释计算器

计算配制储备溶液所需的稀释度。

浓度1（C1）

x

体积 1 (V1)

=

浓度 2 (C2)

x

体积 2 (V2)

复溶计算器

复溶计算器允许您快速计算试剂的体积，以复溶小瓶。只需输入试剂的质量和目标浓度，计算器将确定其余部分。

体积（添加到小瓶中）

=

质量（小瓶）

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所需的复溶浓度

哺乳动物蛋白抽提试剂盒

库存信息