接着および非接着性の哺乳動物細胞の in situ細胞下分画で
Summary
顕微鏡のカバーグラス上に、哺乳動物細胞の in situ細胞下分画ではタンパク質の局在を可視化できる。
Abstract
Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix.
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.
Protocol
Discussion
一般的な問題や提案：
すべてまたは細胞のほとんどは、洗浄中に失われます。洗浄ステップ中に液体がカバースリップを避ける6ウェルプレートの側に徐々に追加する必要があります。同様に液体を慎重に傾けプレートによって除去されるべきであると徐々に余分な水分をオフにピペッティング。細胞接着は、ポリ- L -リジンコートしたカバースリップを使用して増やすこ?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Anyaporn SawasdichaiとNazefahアブドゥルハミドは、博士号のためにそれぞれタイ王国政府とマレーシア政府に感謝しています奨学金。この作品は、PSJとKGに授与ウェルカムトラストのプロジェクトの助成金によって賄われていた。我々はまた、ブリストルウォルフソンのバイオイメージング施設の大学に感謝しています。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| BSA | Chemical | Sigma | A9647-100G | |
| DNase I | Chemical | Sigma | DN25-1G | |
| poly-L-Lysine | Chemical | Sigma | P-8920 | |
| Trypsin | Chemical | |||
| EGTA | Chemical | Sigma | E4378-25G | |
| Fomaldehyde | Chemical | BDH | 284216N | |
| PIPES | Chemical | Sigma | P-6757 | |
| Sucrose | Chemical | Fisher Scientific | S/8600/53 | |
| Triton X-100 | Chemical | Sigma | T-6876 | |
| Mounting Medium with DAPI | Chemical | Vector Laboratories | H-1200 | |
| Fugene 6 Transfection Reagent | Chemical | Roche | 11814443001 | |
| Histone H1 | Antibodies | Santa Cruz | SC-8030 | |
| Lamin A/C | Antibodies | Santa Cruz | SC-20681 | |
| Tubulin-α | Antibodies | Santa Cruz | SC-32293 |
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