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ただし、半自動のアプローチはまた通常使用、例えば急速な採用 など ビデオ顕微鏡低倍率でまたはセル固定および汚損による数百細胞の注入効果の分析に適しています。採用詳細な手法に関係なくマイクロインジェクション エンドポイント アッセイを構成していませんが、さまざまなテクニック、FRAP または photoactivation の 3 を含むと組み合わせることができます。.
PA GFP 付けられた構造の photoactivation のための最適な条件を設定する場合 photoactivation の中にインスタント漂白を避けるために注意が必要があります。レーザーの力で我々 の仕事で最良の結果が得られた 倍低い EGFP の漂白のため通常用いられる。センサー分子の画像を取得する露光時間とフレーム間の時間間隔が地域とセンサー構造のサイズを考慮して最適化して分析する、センサーの潜在的な移動だけでなく、蛋白質は細胞内の他の場所に。蛍光イメージングのすべての種類、細胞生存率の維持は生理学的に関連する結果を取得するため重要です。.
次のダイナミクスの均等に強力なメソッドと、lamellipodium など細胞レベル下の構造の売り上げ高は、原則として mEos など質のバリエーション 12 蛍光タンパク質の緑-赤光変換を構成する 例えば、 見なさい・ バーネット ら PA GFP ではなく後者の方法の利点は前に、と共同その他の赤い蛍光蛋白質を表現する必要性なし、2 つの異なる色で変換後タンパク質ダイナミクスに続く可能性であります。ただし、予備実験でコントラストの変化の程度、PA GFP の photoactivation の時に達成した蛍光信号の強さが大きかった photoconverted プローブと比較しておそらく赤と緑の優れた分光機能のため蛍光プローブ データは示されていない 。いずれの場合では、詳細な研究ケラトサイトやワクシニア ウイルス性アクチン尾などセル端の突起部にアクチン フィラメントの売り上げ高のところのみ公開されている PA GFP 誘導体 5 , 6 , 24 を使用しています。.
次の photoactivation を分析するどのセル領域を考慮する場合いくつかの要因べきである考慮し、 細胞質で活性化セルの端でのアクチン単量体の定款 を示す具体的な例を使用して説明するが、様々 な類似している科学的な問題に確かに推定することができます。まず、測定時葉状 cytosolically センサー蛋白質、例えば、定款の異なる実験条件で ディムチェフ ら に示すよう 6 ゾル性細胞質領域と葉状の端に彼らの間隔のサイズは、実験群間で同等にする必要があります。また、photoactivating 細胞質領域セル厚が核に近い位置で大きいときそれを考慮することが重要です。厚い細胞領域をアクティブにすると、与えられたがアクティブにする蛋白質の分布、細胞質に分散してゆく活性タンパク質の多量可能性があります。最後に、個々 のセルでアクティブにする蛋白質の表現のレベルを非常に可変することができますは確かに。これら変動の考慮による cytosolically 活性化タンパク質他の起動時に特定の地域で得られた合計の蛍光に対する細胞の混入レベルを比較することが重要です。.
FRAP や光など、他の試金による microinjected 細胞の様々 なパラメーターを分析できます。細胞内動態とアクチン単量体の移動を調査するため、縛ると光を活用する方法を説明しました。FRAP は私たちのグループによって使用されています以前 5 ケラトサイト VASP、Abi、cortactin コフィリンなどにローカライズしてキャッピングの蛋白質、蛋白質の成分存在下における焦点接着斑の回転を解明するための売り上げ高を調査するにはRac の不在通知 4. 明らかに、蛋白質はモノマー、ダイマー、または少しでもオリゴマーとして動いているかどうか、彼らは追加の結合パートナーとの組み合わせで移動かどうか photoactivation、FRAP を区別できません。その種の情報は、蛍光相関分光技術 29 あるいは 30 FLIM フレットから代わりに取得できます。それにもかかわらず、FRAP と光は、直接細胞、関心、細胞内の場所、または研究細胞型のタンパク質に関係なくローカルおよびグローバルなタンパク質の動態を評価するために簡単な方法を構成します。.
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