BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
膜蛋白在细胞中扮演着重要的角色：例如被熟知的识别、信号转导、运输等功能，也是用药的理想的靶点。所得的膜蛋白可以应用于 WB, Dot blot, IP 等下游实验。总膜蛋白提取试剂盒提供简单、快速和可复制的方法，从新鲜的哺乳动物组织和培养细胞中提取总膜蛋白，提取的蛋白纯度高且保持天然活性，绝少交叉污染。该方法基于温度依赖性分离。样品首先在膜蛋白提取缓冲液中混合并匀浆。膜蛋白提取缓冲液在低温（4℃）的时候溶解度高，根据有机物相似相溶的原理，这时膜蛋白就会和提取缓冲液相溶（膜蛋白是嵌在膜中的，水溶性不好）；在 37℃反而溶解度低，从而将大部分提取缓冲液连同膜蛋白一起析出分在下层有机相。
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自备耗材
涡旋仪、离心管
细胞刮刀
可调节式移液枪及枪头
磷酸盐缓冲液(PBS)、去离子水
匀浆器（如果是组织样本）
试剂准备
提取缓冲液 (1×)：使用前，用去离子水稀释成1×提取缓冲液，立即加入 10 µL 蛋白酶抑制剂 (100×)到1mL1×提取缓冲液中，置于冰上；2-8℃保存。
洗涤缓冲液 (1×)：使用前，用去离子水稀释成1×洗涤缓冲液，立即加入 10 µL 蛋白酶抑制剂 (100×)到 1 mL 1×洗涤缓冲液中，置于冰上； 2-8℃保存。
蛋白酶抑制剂 (100×)：即用型；使用前，置于冰上；-20℃保存。用不完的试剂-20℃分装保存，避免反复冻融。
实验步骤
注意：所有的实验步骤需在2-8℃进行。使用预冷的缓冲液和设备。确保所有的溶液是解冻和均匀的。
I 细胞培养物的制备
1. 对于贴壁细胞，用细胞刮刀收获 1-3×10⁷个细胞，然后 500 g 离心 5 min。对于悬浮细胞，500 g 离心 5 min。
2. 用预冷的 PBS 重悬细胞沉淀，500 g 离心 2-3 min，弃上清。
3. 向细胞沉淀中加入 1 mL 预冷的 1× 提取缓冲液后，用匀浆器匀浆细胞 30-50 次，继续步骤 III。
II 组织制备
1. 将 50-100 mg 组织切成小块，放入离心管中。
2. 用预冷的 PBS 清洗组织，500 g 离心 5 min，弃上清。
3. 用 1 mL 预冷的 1×提取缓冲液轻柔重悬组织。
4. 使用匀浆器或组织研磨机匀浆组织，直至超过90%的细胞被破坏，细胞核在显微镜下可见，继续步骤 III。
III 膜疏水和亲水蛋白的分离
1. 将裂解物放置于冰上10 min。
注意：如果有需要，可以保存 50-100 μL 总蛋白质裂解物，做进一步分析。
2. 10,000 g，4℃离心 5 min。
3. 将上清液转移到新的离心管中并弃掉沉淀，上清在37℃孵育 5 min，直至变浑浊，在孵育期间，上下颠倒离心管。
4. 3,000 g，室温离心 3 min。 注意：确保离心机的温度高于20℃，不要将离心后的管放到冰上。离心后注意观察液体分层，离心后的溶液必须有明显分层，否则重复该步骤。上层液体为亲水相（胞浆蛋白），下层呈粘稠油状为疏水相，含疏水蛋白（膜蛋白）。
5. 下层疏水相极大的富集了疏水蛋白，将上层包含亲水蛋白的亲水相转移到新的离心管做进一步分析。
6. 为了从疏水相中去除残留的亲水蛋白，加入 400 μL 1×洗涤缓冲液到疏水相，轻柔混匀，冰上孵育 10 min，重复步骤 3-5。
7. 如果无需立即进行下游实验，分装膜蛋白，并迅速冷冻后，储存于-80℃。
8. 对于SDS-PAGE：直接使用分离的膜蛋白煮沸后进行电泳。对于IP实验：在加入抗体前，可用 10-15倍的1×洗涤缓冲液先稀释疏水蛋白。
注意：如果需要，可以通过 TCA 沉淀样品以获得更浓缩的样品。
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Consumables to be prepared by users
Vortex mixer, centrifuge tubes
Cell scraper
Adjustable pipettes and pipette tips
Phosphate Buffered Saline (PBS), deionized water
Homogenizer (for tissue samples only)
Reagent Preparation
1× Extraction Buffer: Before use, dilute the stock solution to 1× working concentration with deionized water. Immediately add 10 μL of 100× Protease Inhibitor Cocktail to every 1 mL of 1× Extraction Buffer, place on ice and store at 2-8 °C.
1× Wash Buffer: Before use, dilute the stock solution to 1× working concentration with deionized water. Immediately add 10 μL of 100× Protease Inhibitor Cocktail to every 1 mL of 1× Wash Buffer, place on ice and store at 2-8 °C.
100× Protease Inhibitor Cocktail: Ready-to-use formulation. Keep on ice prior to use; long-term storage at −20 °C. Aliquot remaining reagents and store at −20 °C to prevent repeated freeze-thaw cycles.
Experimental Procedures
Note: All operations must be performed at 2–8 °C. Use pre-cooled buffers and equipment. Ensure all solutions are fully thawed and well homogenized.
I. Preparation of Cultured Cells
1.For adherent cells: Harvest 1–3×10⁷ cells with a cell scraper, then centrifuge at 500 g for 5 min. For suspension cells: centrifuge directly at 500 g for 5 min.
2.Resuspend the cell pellet with pre-cooled PBS, centrifuge at 500 g for 2–3 min, and discard the supernatant.
3.Add 1 mL pre-cooled 1× Extraction Buffer to the cell pellet, homogenize 30–50 times with a homogenizer, then proceed to Step III.
II. Tissue Preparation
1.Cut 50–100 mg fresh tissue into small pieces and transfer into a centrifuge tube.
2.Wash the tissue with pre-cooled PBS, centrifuge at 500 g for 5 min, and discard the supernatant.
3.Gently resuspend the tissue in 1 mL pre-cooled 1× Extraction Buffer.
4.Homogenize the tissue with a homogenizer or tissue grinder until over 90% of cells are disrupted and intact nuclei can be observed under a microscope, then proceed to Step III.
III. Separation of Hydrophobic Membrane Proteins and Hydrophilic Proteins
1.Incubate the lysate on ice for 10 min.
Note: Reserve 50–100 μL total protein lysate for subsequent analysis if required.
2.Centrifuge at 10,000 g, 4 °C for 5 min.
3.Transfer the supernatant into a new centrifuge tube and discard the pellet. Incubate the supernatant at 37 °C for 5 min until turbidity appears, and invert the tube upside down several times during incubation.
4.Centrifuge at 3,000 g at room temperature for 3 min.
Note: Confirm the centrifuge chamber temperature is above 20 °C; do not place centrifuged tubes on ice immediately after centrifugation. Distinct liquid layers must be visible after centrifugation, otherwise repeat this step. The upper layer is the hydrophilic phase (cytosolic proteins), and the viscous oily lower layer is the hydrophobic phase enriched with hydrophobic membrane proteins.
5.The lower hydrophobic phase is highly enriched in hydrophobic membrane proteins. Transfer the upper hydrophilic phase containing cytosolic proteins to a new tube for further testing.
6.To remove residual hydrophilic proteins from the hydrophobic phase, add 400 μL 1× Wash Buffer, mix gently, incubate on ice for 10 min, then repeat Steps 3 to 5.
7.If downstream experiments cannot be carried out immediately, aliquot the membrane protein samples, snap-freeze quickly and store at −80 °C.
8.For SDS-PAGE: Boil the isolated membrane proteins directly before electrophoresis. For IP assays: Dilute the hydrophobic protein fraction with 10–15 volumes of 1× Wash Buffer prior to antibody incubation.
Additional Note: TCA precipitation can be applied to further concentrate samples if needed.
| 组分货号 | 组分名称 | 物理外观 | 50T | 200T | 储存 |
| T1526926A | 提取缓冲液（2×） | 液体 | 25 mL | 100 mL | 2-8℃. |
| T1526926B | 洗涤缓冲液（2×） | 液体 | 15 mL | 60 mL | 2-8℃. |
| T1526926C | 蛋白酶抑制剂（100×） | 液体 | 0.8 mL | 3.2 mL | -20℃ |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0939444 |
分析证书 | T1526926 |
| ZJ26F0939443 |
分析证书 | T1526926 |
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