太鼎生物科技/太鼎食安科技

細胞內蛋白質樣品萃取與製備

線粒體/細胞質分離試劑盒

Mitochondria/Cytosol Fractionation Kit
Catalog#: K256-100 

K256-25 Mitochondrial/Cytosol Fractionation Kit 25 assays

K256-100 Mitochondrial/Cytosol Fractionation Kit 100 assays

該試劑盒用於分離凋亡或非凋亡的哺乳動物細胞的細胞質和線粒體，可進行凋亡研究，或用Western Blot、ELISA來檢測，目標蛋白於細胞質和線粒體的localization情況。

Kit Summary:
• Detection method- Western blotting, ELISA, or other assays.
• Sample type- Cells and Tissues
• Species reactivity- Mammalian
• Kit size- 25, 100 assays
• Applications- Effective isolation of a highly enriched mitochondrial fraction from cytosolic fraction of mammalian cells including both apoptotic and nonapoptotic cells.

Features & Benefits:
• Simple procedure; takes only 3-4 hours
• Fast and convenient
• The fractionation procedure is simple and staightforward. No ultracentrifugations are required. No toxic chemicals are involved.

Kit components:
• Mitochondria Extraction Buffer
• 5X Cytosol Extraction Buffer
• DTT ( 1 M )
• Protease Inhibitor Cocktail

Description:
The Mitochondria/Cytosol Fractionation Kit provides unique formulations of reagents for effective isolation of a highly enriched mitochondrial fraction from cytosolic fraction of mammalian cells including both apoptotic and nonapoptotic cells. The enriched mitochondrial and cytosolic fractions can be used for studying apoptotic and signal transduction pathways to detect translocation of factors interested between the two fractions by Western blotting, ELISA, or other assays. Procedures are simple and easy to perform, no ultracentrifugations and toxic chemicals are involved.

細胞核/細胞質分離試劑盒

Nuclear/Cytosol Fractionation Kit
Catalog#: K266-100  

K266-25 Nuclear/Cytosol Fractionation Kit 25 assays

K266-100 Nuclear/Cytosol Fractionation Kit 100 assays

試劑盒內包含，分離哺乳動物細胞的細胞核和細胞質所有試劑，不僅能夠分離細胞核和細胞質蛋白質，而且可以保持蛋白質的完整性。所分離的細胞核和細胞質蛋白質可進行轉錄活性、RNA 剪接、凝膠阻滯電泳實驗、報告蛋白檢測、酶活性檢測及Western Blot 研究等。

Kit Summary:
• Sample type- Cell culture (adherent and suspension), fresh and frozen tissues
• Species reactivity- Mammalian
• Kit size- Conveient sizes (25, 100)
• Applications- This Nuclear/Cytosol Extraction Kit provides a complete system that enables the separation of nuclear extract from the cytoplasmic fraction of mammalian cells and tissues The new kit provides a system that maintains the nuclear and cytoplasmic compartments separate and intact. Transcriptional activity/reporter assays/enzyme activity assays/Western blotting and more downstream assays.

Features & Benefits:
• Simple procedure; takes less than 2-3 hours
• Fast and convenient
• The optimized reagents and procedures provided with the kit allow separation of nuclear and cytoplasmic extracts from cells relatively quick with little or no cross-contaminations

Kit components:
• Cytosol Extraction Buffer A (CEB-A)
• Cytosol Extraction Buffer B (CEB-B)
• Nuclear Extraction Buffer (NEB)
• DTT ( 1 M )
• Protease Inhibitor Cocktail (lyophilized)

Description:
This Nuclear/Cytosol Extraction Kit provides a complete system that enables the separation of nuclear extract from the cytoplasmic fraction of mammalian cells. The optimized reagents and procedures provided with the kit allow separation of nuclear and cytoplasmic fractions quickly with little or no cross-contaminations. The extracted nuclear and cytoplasmic protein fractions are functional and compatible with downstream assays such as transcriptional activity, RNA splicing, gel shift assay, reporter assays, enzyme activity assays, and Western blotting.

細胞質/胞內顆粒快速分離試劑盒

K267-50 Cytosol/Particulate Rapid Separation Kit 50 assays

試劑盒利用物理學原理，通過一個油層將細胞質和胞內顆粒快速分開，使得兩個蛋白質不能互相接觸或彌散，進而實現對蛋白質進行精確的亞細胞定位和分析。

哺乳細胞 / 膜蛋白抽提試劑盒

Membrane Protein Extraction Kit
Catalog#: K268-50  

K269-500 Mammalian Cell Extraction Kit 500 assays

K268-50 Membrane Protein Extraction Kit 50 assays

該試劑盒提供最佳化的緩衝液和試劑，用來高效地萃取哺乳動物組織和細胞的膜蛋白。傳統的方法或試劑盒僅能提取總的細胞膜蛋白，包括漿膜和細胞器膜蛋白。該試劑盒不僅能分離總的細胞膜蛋白，還可以單獨純化漿膜蛋白(plasma membrane protein)。試劑盒所抽提出的膜蛋白可用於Western Blot，二維電泳和酶活性分析等多種檢測方法。

Kit Summary:
• Sample type- Cell culture (adherent and suspension), fresh and frozen tissues
• Species reactivity- Mammalian
• Applications- Western blotting, 2-D gels, and enzyme analyses, etc. The entire procedure takes less than 1 hour.

Features & Benefits:
• Simple procedure
• Fast and convenient
• The Membrance Protein Extraction Kit provides an easy and complete protocol for isolating integral membrance proteins efficiently from cultured mammalian cells.

Kit components:
• Homogenize Buffer
• Upper Phase Solution
• Lower Phase Solution
• Protease Inhibitor Cocktail

Description:
The Membrane Protein Extraction Kit provides optimized buffers and reagents for effective extraction of membrane proteins from mammalian tissues and cells. Unlike other available procedures that can only extract the total cellular membrane proteins (combinations of plasma and organelle membrane proteins), BioVision’s kit was designed to not only extract the total cellular membrane proteins, but also purify the plasma membrane proteins specifically. The procedure offers consistent yield and high purity (over 90%). Membrane proteins prepared using the kit can be utilized in a variety of applications, such as Western blotting, 2-D gels, and enzyme analyses, etc. The entire procedure takes less than 1 hour.

四合一胞器分離試劑盒

FractionPREP™ Cell Fractionation Kit

K270-50 Fraction-PREPTM Cell Fractionation System 50 assays

該試劑盒能從一個哺乳動物樣本中抽提出四種蛋白質，細胞質、細胞核、膜/顆粒及細胞骨架組分等。方法簡單、快速，整個過程只需2 小時；無需超速離心；結果重複性好； 所分離的蛋白質可用於一維或二維電泳。

 K270-50 FractionPREP™ Cell Fractionation Kit 細胞分離套組：

• 快速：2小時內快速萃取細胞質、細胞膜、細胞核、細胞骨架等細胞內蛋白質
• 方便：Ready-to use試劑，不需超高速離心。
• 種類：細胞、組織皆適用
• 應用：適用於各類蛋白質分析實驗。如SDS-PAGE、2D Gel electrophoresis、Western blot、ELISA、EMSA、RNA splicing、translocation、reporter assays、酵素活性分析等。 

Kit Summary:
• Sample type- Cell culture (adherent and suspension), fresh and frozen tissues
• Species reactivity- Mammalian
• Applications- 1-D or 2-D gel, enzyme activity assays, gel shift assay, and Western blotting.

Features & Benefits:
• Simple procedure; takes less than 1 hour
• Fast and convenient
• The kit is designed for serial sample preparation of four distinct protein fractions including cytosol/particulate/cytoskeleton/nuclear fractions, from one sample.

Kit components:
• Cytosol Extraction Buffer (CEB)
• Membrane Extraction Buffer-A (MEB-A)
• Membrane Extraction Buffer-B (MEB-B)
• Nuclear Extraction Buffer (NEB)
• DTT ( 1 M )
• Protease Inhibitor Cocktail (lyophilized)

Description:
This FractionPREP™ Cell Fractionation system is designed to provide reproducible extraction of four subcellular protein fractions (Cytosol, nucleus, membrane/particulate, and cytoskeletal fractions) from a single mammalian sample. The method is fast and simple, needing only 2 hours and no ultracentrifugation involved. All four protein fractions obtained are suitable for many downstream applications such as 1-D or 2-D gel, enzyme activity assays, gel shift assay, and Western blotting

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胞器蛋白分離的應用(一)

目的: 探討細胞核內轉錄因子和特定DNA序列相互作用

應用: 檢測蛋白質和一段特定DNA序列(label)相互結合的技術

原理: 當特定序列的DNA和某蛋白質結合後，整個蛋白質複合體分子量會增加,經由電泳後,在gel中的移動速度變慢,整個band進上shift.

可加入特定抗體檢測是否有特定蛋白質Binding( Supershift)

胞器蛋白分離的應用(二)

應用: 細胞凋亡時,粒線體膜電位去極化的過程會增加膜的通透性並使膜內蛋白,如Cytochrome C, AIF, Smac/DIABLO等分子釋放到細胞質中,引發caspase-9活化, 造成一連串凋亡反應發生. COX4蛋白位於粒腺體細胞內側, 並在Apoptosis發生時依舊保留在粒線體內。

胞器蛋白分離的應用(三)

應用: 二維電泳與MDLC(液相層析)並列為研究蛋白質體學的重要技術之一

原理: 利用分子量(一維)與等電點(二維)一次可分離大約2000個蛋白質

限制: 膜蛋白(Hydrophobic)在電泳中不易看到.蛋白質若是為low abundant protein則欲得到準確而可信的實驗結果要做胞器分離( sample prefractionation) 胞器分離在2d應用的優點:

         1. 移去abundant protein 

         2. 增加 low abundant protein

         3. 減少蛋白質的複合體發生

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胞器蛋白分離的操作方式

以FractionPREP Cell Fractionation Kit的操作方法為例:其它組kit方法雷同

操作方法

1. 收集4~8x106~400mg組織/1ml PBS,700xg離心5分, 以5~10ml的預冷PBS wash,後以700xg再離心5分

2. 將細胞重新懸浮於1ml的預冷PBS,700xg 離心5分,並移去supernatant.

3. 加入400ul cytosol extraction buffer-mix(已加DTT及Protease inhibitor cocktail) pipette數次以混合細胞

4. 冰上放置20分鐘作用, 每5分翻轉3~4次

5. 將sample700xg離心10分鐘, 並收集supernatant----

    à所得到為cytosolic fraction

6. 將留下的pellet再加入400ul 預冷的membrane extraction buffer A mix,  pipetter 10~15秒以混合均勻

7. 加入22ul的membrance extraction buffer-B 振盪5秒後冰上靜置1分

8. 再振盪5秒後以1000xg離心5分,收集supernatatn於預冷的tube

    à所得為membrane/partivulate fraction

9. 將pellet再加入200ul預冷的Nuclear extraction buffer mix, 振盪15秒後,冰上放置40分,每10分振盪15秒

10. 高速離心10分, 將supernatant 轉移至新的預冷之tube—

    à所得為nuclear fraction

11. 最後所留下的pellet,再溶於100ul buffer(0.2% SDS, 10mM DTT)irm SDS-PAGE sample buffer)

    à所得為cytoskeletal fraction

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