本试剂盒创新性地采用过柱纯化的方法，能够快速、温和、高效地裂解动物组织或细胞，有效提取总蛋白。试剂盒同时提供变性和天然两种裂解液，用户可根据下游实验需求进行选择。整个提取过程仅需要1~8 min，由于采用过柱纯化技术，最小可处理20μL样本与裂解液混合物，最大可达500μL，提取的蛋白溶液浓度可达2~8mg/mL，并可有效避免蛋白丢失。所提蛋白可采用BCA法进行蛋白定量分析（货号：R1491648/B665595）。
C1491674
| Component
| 50T | Storage
|
C1491674A
| 变性裂解液
| 25 mL | 2-8℃ |
C1491674B
| 天然裂解液
| 25 mL
| 2-8℃
|
C1491674C
| 纯化柱
| 50个 | RT. |
C1491674D
| 收集管
| 50个
| RT.
|
C1491674E
| 塑料研磨棒
| 4根 | RT. |
产品特点
1、操作简单快速，最快1min即可得到变性总蛋白；
2、无蛋白丢失，可打开DNA双链，高效获取与DNA结合的蛋白；
3、小样本量、高得率，最小可处理20μL样本与裂解液混合物，提取的蛋白溶液浓度可达2~8mg/mL；
4、适用多种实验，含有两种裂解液，既可用于提取变性蛋白质，也可提取天然蛋白质。
使用说明
一、提取变性总蛋白
1、将纯化柱及收集管放在冰上预冷；
2、样品处理（取适当量的变性裂解液，在使用前数分钟将蛋白酶抑制剂混合液按 1:100 加入其中；
2.1 贴壁细胞：将预冷的1×PBS 直接加入培养板、培养皿或培养瓶中清洗贴壁细胞，吸去上清。按照附表（文末）中将相应体积的变性裂解液均匀地加入整个器皿表面，用移液器吹打几次。
2.2 悬浮细胞：低速离心收集细胞，在1.5mL离心管中加入预冷的1×PBS，涡旋震荡，3,000rpm离心2~3min清洗细胞。吸去多余上清，留下与细胞相同体积的PBS，涡旋震荡重悬细胞。加入附表中相应体积的变性裂解液 ，涡旋震荡裂解细胞。
注意：
① 部分未完全裂解的细胞不会影响后续蛋白提取效果；
② 加入裂解液后，如果细胞裂解物太过粘稠，无法用200~1,000μL吸头吹打，可将细胞裂解物直接倒入纯化柱中，进行后续操作。
2.3 组织样本：将15~20 mg组织放置于纯化柱上，用塑料研磨棒扭转研磨50~60次，加入200 μL 变性裂解液，继续研磨30~60次。如样本起始量较大或者较小，需按比例调整相应裂解液的用量；
注意：塑料研磨棒可以重复使用，请用蒸馏水彻底冲洗干净，并用纸巾擦干。
3、离心
3.1 贴壁细胞或悬浮细胞：将裂解后的细胞转移到预冷的纯化柱套管中，14,000~16,000 rpm离心 30 s取出；
3.2 组织样本：盖上纯化柱盖子室温孵育1~2 min，14,000~16,000 rpm离心1~2 min取出。
4、立刻将收集管放置于冰上，弃去纯化柱，变性总蛋白提取完成。
二、提取天然总蛋白
1、将天然裂解液、纯化柱及收集管放在冰上预冷；
2、样品处理（取适当量的天然裂解液，在使用前数分钟将蛋白酶抑制剂混合液按 1:100 加入其中；
2.1 贴壁细胞：将预冷的1×PBS直接加入培养板，培养皿或培养瓶中清洗贴壁细胞，吸去上清。按照附表中将相应体积的天然裂解液均匀地加入整个器皿表面，放置于冰上孵育 3~5 min，用移液器吹打几次；
2.2 悬浮细胞：低速离心收集细胞，在1.5 mL离心管中加入预冷的1×PBS，涡旋震荡，3,000 rpm离心2~3 min清洗细胞。吸去多余上清，留下与细胞相同体积的PBS，涡旋震荡重悬细胞。加入附表中相应体积的天然裂解液，涡旋震荡裂解细胞15s。将离心管放置于冰上3~5 min，然后涡旋震荡10s；
注意：
① 部分未完全裂解的细胞不会影响后续蛋白提取效果；
② 加入裂解液后，如果细胞裂解物太过粘稠，无法用200~1,000 µL吸头吹打，可将细胞裂解物直接倒入纯化柱中，进行后续操作。
2.3 组织样本：将15~20 mg组织放置于纯化柱上，用塑料研磨棒扭转研磨50~60次，加入200 μL 天然裂解液，继续研磨30~60次。如样本起始量较大或者较小，需按比例调整相应裂解液的用量；
注意：塑料研磨棒可以重复使用，请用蒸馏水彻底冲洗干净，并用纸巾擦干。
3、离心
3.1 贴壁细胞或悬浮细胞：将裂解后的细胞转移到预冷的纯化柱套管中，14,000~16,000 rpm离心 30 s 取出；
3.2 组织样本：开盖冰上孵育5 min，盖上纯化柱盖子，4℃，14,000~16,000 rpm离心1~2 min取出；
4、立刻将收集管放置于冰上，弃去纯化柱，天然总蛋白提取完成。
附表 细胞数量与所需裂解液体积之间的关系
注意事项
1、若使用本试剂盒裂解液裂解样本所得产物比较粘稠，此为正常现象；
2、为了您的安全和健康，请穿实验服并戴一次性手套操作；
3、本产品仅限科研使用。
物理外观：
C1491674A, C1491674B: Liquid
This kit innovatively adopts a column-based purification method to rapidly, gently, and efficiently lyse animal tissues or cells for total protein extraction. It provides both denaturing and native lysis buffers, allowing users to select the appropriate option based on downstream application requirements. The entire extraction process takes only 1–8 minutes. Thanks to the column purification technology, it can process sample-lysis buffer mixtures as small as 20 μL and up to 500 μL, yielding protein solutions with concentrations of 2–8 mg/mL while effectively preventing protein loss. The extracted proteins can be quantified using the BCA method (Cat. No.: R1491648/B665595).
C1491674
| Component
| 50T | Storage
|
C1491674A
| Denaturing Lysis Buffer
| 25 mL | 2-8℃ |
C1491674B
| Native Lysis Buffer
| 25 mL
| 2-8℃
|
C1491674C
| Purification Columns
| 50 units | RT. |
C1491674D
| Collection Tubes
| 50 units
| RT.
|
C1491674E
| Plastic Grinding Pestles
| 4 units | RT. |
Key Features
1.Simple and rapid operation: Denatured total proteins can be obtained in as little as 1 minute.
2.No protein loss: Efficiently extracts DNA-binding proteins by disrupting DNA duplexes.
3.Small sample volume, high yield: Processes mixtures as small as 20 μL, yielding protein concentrations of 2–8 mg/mL.
4.Versatile applications: Includes two lysis buffers for extracting both denatured and native proteins.
Protocol
I. Extraction of Denatured Total Protein
1.Pre-chill the purification column and collection tube on ice.
2.Sample processing: Add protease inhibitor cocktail to the denaturing lysis buffer at a 1:100 ratio shortly before use.
2.1 Adherent cells:
Wash cells with pre-chilled 1× PBS and aspirate the supernatant.
Add the volume of denaturing lysis buffer specified in the appendix table to cover the culture surface, and pipette to mix.
2.2 Suspension cells:
Collect cells by low-speed centrifugation.
Wash with pre-chilled 1× PBS, vortex, and centrifuge at 3,000 rpm for 2–3 minutes.
Resuspend the cell pellet in PBS equal to the pellet volume.
Add the specified volume of denaturing lysis buffer and vortex to lyse.
Note: Partial incomplete lysis does not affect protein extraction. If the lysate is too viscous, directly transfer it to the purification column.
2.3 Tissue samples:
Place 15–20 mg of tissue on the purification column.
Grind 50–60 times with a plastic pestle.
Add 200 μL denaturing lysis buffer and grind another 30–60 times.
Adjust lysis buffer volume proportionally for larger or smaller samples.
Note: Reusable plastic pestles should be thoroughly rinsed with distilled water and dried.
3.Centrifugation:
3.1 Adherent or suspension cells: Transfer the lysate to the pre-chilled purification column and centrifuge at 14,000–16,000 rpm for 30 seconds.
3.2 Tissue samples: Incubate the column at room temperature for 1–2 minutes, then centrifuge at 14,000–16,000 rpm for 1–2 minutes.
4.Immediately place the collection tube on ice and discard the purification column. Denatured total protein extraction is complete.
II. Extraction of Native Total Protein
1.Pre-chill the native lysis buffer, purification column, and collection tube on ice.
2.Sample processing: Add protease inhibitor cocktail to the native lysis buffer at a 1:100 ratio shortly before use.
2.1 Adherent cells:
Wash cells with pre-chilled 1× PBS and aspirate the supernatant.
Add the specified volume of native lysis buffer and incubate on ice for 3–5 minutes. Pipette to mix.
2.2 Suspension cells:
Collect, wash, and resuspend cells as described in section I.
Add native lysis buffer, vortex for 15 seconds, incubate on ice for 3–5 minutes, and vortex again for 10 seconds.
2.3 Tissue samples:
Grind tissue as described in section I, using native lysis buffer.
3.Centrifugation:
3.1 Adherent or suspension cells: Centrifuge at 14,000–16,000 rpm for 30 seconds.
3.2 Tissue samples: Incubate on ice for 5 minutes (open lid), then close the lid and centrifuge at 4°C and 14,000–16,000 rpm for 1–2 minutes.
4.Immediately place the collection tube on ice and discard the purification column. Native total protein extraction is complete.
Cell Count (×10⁶)
| Lysis Buffer Volume (μL)
|
| 0.3 | 20 |
| 0.5 | 50 |
| 1 | 100 |
| 2 | 200 |
| 3 | 500 |
Appendix: Cell Number vs. Lysis Buffer Volume
Precautions
1.High viscosity of the lysate is normal when using this kit.
2.For safety, wear a lab coat and disposable gloves during operation.
3.For research use only.
ZJ25F0927265
危险品化学品经营许可证（带存储）