細胞核/細胞質分離試劑盒 | Nuclear/Cytosol Fractionation Kit貨號KTP3001

細胞核提取物的製備。所分離的細胞核和細胞質蛋白質可進行轉錄活性(transcriptional activity)、RNA 剪接 (RNA splicing)、電泳遷移率變動分析（Electrophoresis Mobility Shift Assay , EMSA）、足跡分析(footprinting analysis)、報告蛋白檢測 ( reporter assays)、轉錄分析(transcription assays)、酶活性檢測 (enzyme activity assays)、 純化調節蛋白(regulatory proteins)的起點、蛋白質印跡(Western Blot )研究等。

 

Fig.1. Western blot of specific proteins fraction using Abbkine ExKine™ Nuclear and Cytoplasmic Protein Extraction Kit. Sample: 293T cells lysate. C: cytoplasmic extraction was analyzed using α-tubulin antibody (A01080). N: nuclear extraction was analyzed using α-tubulin antibody (A01080).Fig.2. Western blot of specific proteins fraction using Abbkine ExKine™ Nuclear and Cytoplasmic Protein Extraction Kit. Sample: 293T cells lysate. C: cytoplasmic extraction was analyzed using Histone H3 antibody (A01070). N: nuclear extraction was analyzed using Histone H3 antibody (A01070).

產品介紹:

Nuclear/Cytosol Extraction Kit試劑盒內包含，分離哺乳動物細胞/組織之細胞核和細胞質所有試劑，也包括Protease Inhibitor Cocktail 100X。該試劑盒應用原理基於細胞在低滲緩衝液中溶脹的功能。然後是細胞被破壞，去除細胞質部分，釋放核蛋白通過高鹽緩衝液從細胞核中分離出來。非變性的活性蛋白可在短時間內純化。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。 2×10⁶ cells 或30~60 mg組織/反應。可應用於哺乳動物培養細胞或組織樣品。

細胞核/細胞質分離試劑盒KTP3001操作

1. Vortex the tube vigorously on the highest setting for 15 seconds to fully suspend the cell
2. Add 10 uL ice-cold CES B to the tube. Vortex the tube for 10-15 seconds on the highest setting. Incubate tube on ice for 1–2 minute.
3. Centrifuge the tube at 16000 g for 5 minutes at 4 °C.
4. Immediately transfer the supernatant (cytoplasmic extract) to a clean pre-chilled tube. Place this tube on ice until use or at -80°C for longer storage. The pellet (which contains nuclei) is usually viscous and not very compact. Optional: In order to remove residual cytoplasmic protein from the nuclei, rinse the pellet with additional ice-cold CES A buffer or PBS. And then repeat procedure C Step 3 and 4.
5. Suspend the pellet in 100 uL ice-cold NES. Place the sample on ice and continue vortexing for 15 seconds every 10 minutes, for a total of 30 minutes. Avoid foam formation.
6. Centrifuge at 16000 g for 15 minutes at 4°C.
7. Aliquot supernatant (nuclear extract) to clean chilled tubes, keeping a small aliquot aside for protein quantification, flash freeze and store at -80 oC. Avoid freeze/thaw cycles. Note: The nuclear proteins extracted according to the protocol are suspended in NES, a high salt buffer. If large volumes of nuclear extract are required in subsequent applications or if problems occur with downstream assays, dialyze the nuclear extract to remove excess salts before use.

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胞器蛋白分離的應用

1. 探討細胞核內轉錄因子和特定DNA序列相互作用。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進上可加入特定抗體檢測是否有特定蛋白質Binding( Supershift)
2. 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。
3. 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: 移去abundant protein 、增加 low abundant protein、減少蛋白質的複合體發生