JP6120335B2 - ルシフェラーゼシグナルを増強する組成物 - Google Patents
ルシフェラーゼシグナルを増強する組成物 Download PDFInfo
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- JP6120335B2 JP6120335B2 JP2014218368A JP2014218368A JP6120335B2 JP 6120335 B2 JP6120335 B2 JP 6120335B2 JP 2014218368 A JP2014218368 A JP 2014218368A JP 2014218368 A JP2014218368 A JP 2014218368A JP 6120335 B2 JP6120335 B2 JP 6120335B2
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Description
(a)ルシフェラーゼを発現する細胞を提供するステップであって、ルシフェラーゼが主に又は完全に不活性な状態又はコンホメーションで存在するステップと;
(b)不活性な状態又はコンホメーションから活性な状態又はコンホメーションへとルシフェラーゼを変換することができる有効量の試薬組成物と一緒に細胞をインキュベートするステップと;
(c)ルシフェラーゼ酵素の基質及び必要に応じてルシフェラーゼの生物発光活性に必要なCoA、ATP、マグネシウム等の補因子を添加するステップと;
(d)活性型ルシフェラーゼによって発生した生物発光シグナルを検出するステップと;
を含む方法を提供する。
(a)1又はそれ以上の細胞を溶解するステップと;
(b)細胞溶解物を、キレータを含む有効量の試薬組成物と接触させるステップと;
(c)ルシフェラーゼ酵素の基質及び必要に応じてルシフェラーゼの生物発光活性に必要なCoA、ATP、マグネシウム等の補因子を添加するステップと;
(d)試料中の生物発光を検出するステップと;
を含み、組換えルシフェラーゼが、天然の形態では分泌型のルシフェラーゼの非分泌型変種である、方法を提供する。
(a)試料を有効量の本発明の試薬組成物とインキュベートするステップと;
(b)ルシフェラーゼ基質を添加するステップと;
(c)試料中の生物発光を検出するステップと;
を含む、試料中のルシフェラーゼの量又は活性を決定する方法に関する。
R−SH + R1−SH → R−S−S−R1 + 2H+ + 2e− 1.0
パート1:ルシフェラーゼ
0.1% NP40
150mM NaBr
5% グリセロール
5mM EDTA
25mM トリス pH8.5
63.4μM シュウ酸ナトリウム
0.6mM 還元型グルタチオン
0.4mM 酸化型グルタチオン
23.6μM セレンテラジン
1mM EDTA
25mM トリス pH7.75
0.6mM 還元型グルタチオン
0.4mM 酸化型グルタチオン
2mM アスコルビン酸塩
75mM 尿素
改変された非分泌型ガウシアルシフェラーゼを発現するHeLa細胞を96穴プレートで培養し、培地を全て取り除いた後、溶解バッファ(20μl)で溶解し、Wallac Victor3ルミノメータ(Perkin Elmer社製)を用いて、アッセイバッファ(60μl)注入後のフラッシュ反応におけるルシフェラーゼ活性をアッセイする。
Claims (25)
- ルシフェラーゼに基づくアッセイを行う方法において、
野生型として分泌されるルシフェラーゼを真核細胞中で発現させるステップであって、当該真核細胞中において当該ルシフェラーゼが細胞内で発現するように当該ルシフェラーゼが機能的シグナルペプチドを欠くステップ;
前記真核細胞を可溶化するステップ;
ルシフェラーゼ活性を、前記ルシフェラーゼと、ルシフェラーゼを活性状態へ転換させることを可能とする環境を与える試薬組成物とを接触させることにより回復させるステップであって、当該試薬組成物が、臭化物陰イオン、キレータ、酸化還元バッファ、およびpHが8.0よりも高いバッファからなる群より選択される1つの試薬を含むステップ;
前記ルシフェラーゼとルシフェラーゼ酵素の基質とを接触させるステップ;および
生物発光を検出または測定するステップ;
を具えることを特徴とする方法。 - 請求項1に記載の方法において、活性状態の前記ルシフェラーゼがシステイン残基間にジスルフィド結合を有することを特徴とする方法。
- 請求項1に記載の方法において、前記活性を回復するステップが細胞可溶化過程と同時に起きることを特徴とする方法。
- 請求項1に記載の方法において、前記方法がレポータ遺伝子アッセイの中で用いられることを特徴とする方法。
- ルシフェラーゼに基づくアッセイを行う方法において、
野生型として分泌されるルシフェラーゼを真核細胞中で発現させるステップであって、当該真核細胞中において当該ルシフェラーゼが細胞内で発現するように当該ルシフェラーゼが機能的シグナルペプチドを欠くステップ;
前記真核細胞を可溶化するステップ;
前記ルシフェラーゼと、前記ルシフェラーゼを活性状態へ転換させることを可能とする環境を与える試薬組成物とを接触させるステップであって、当該試薬組成物が、臭化物陰イオン、キレータ、酸化還元バッファ、酸化剤、およびpHが8.0よりも高いバッファの1つまたは2つ以上を含むステップ;
前記ルシフェラーゼとルシフェラーゼ酵素の基質とを接触させるステップ;および
生物発光を検出または測定するステップ;
を具えることを特徴とする方法。 - 請求項5に記載の方法において、活性状態の前記ルシフェラーゼがシステイン残基間にジスルフィド結合を含むことを特徴とする方法。
- 請求項5に記載の方法において、前記ルシフェラーゼと試薬組成物とを接触させるステップが、前記細胞可溶化過程と同時に起きることを特徴とする方法。
- 請求項5に記載の方法において、前記方法がレポータ遺伝子アッセイの中で用いられることを特徴とする方法。
- 請求項5に記載の方法において、前記ルシフェラーゼの活性状態への転換が、不活性状態から活性状態への転換、または、部分的活性または低い活性状態からより高い活性状態への転換を意味することを特徴とする方法。
- サンプル中の組み換えルシフェラーゼ量を検出または定量する方法において、当該方法が:
(a)前記サンプルを、臭化物陰イオン、キレータ、酸化剤、酸化還元バッファ、およびpHが8.0よりも高いバッファの1つまたは2つ以上を含む試薬組成物と接触させるステップ;
(b)前記サンプルを、前記ルシフェラーゼ酵素の基質と接触させるステップ;および
(c)前記サンプル中の生物発光を検出または定量するステップ;
を含み、前記組み換えルシフェラーゼは、機能的シグナルペプチドを欠き、野生型として分泌されるルシフェラーゼの非分泌型変種または誘導体であることを特徴とする方法。 - サンプル中の組み換えルシフェラーゼ量を検出または定量する方法において、当該方法が:
(a)前記サンプルを、前記ルシフェラーゼを活性状態に転換できる環境を与える試薬組成物と接触させるステップであって、当該試薬組成物が臭化物陰イオン、キレータ、酸化剤、酸化還元バッファ、およびpHが8.0よりも高いバッファの1つまたは2つ以上を含むステップ;
(b)前記サンプルを、前記ルシフェラーゼ酵素の基質と接触させるステップ;および
(c)前記サンプル中の生物発光を検出または定量するステップ;
を含み、前記組み換えルシフェラーゼは、機能的シグナルペプチドを欠き、野生型として分泌されるルシフェラーゼの非分泌型変種または誘導体であることを特徴とする方法。 - 請求項11に記載の方法において、前記ルシフェラーゼの活性状態への転換が、不活性状態から活性状態への転換、または、部分的活性または低い活性状態からより高い活性状態への転換を意味することを特徴とする方法。
- 請求項11に記載の方法において、ステップ(b)がステップ(a)と同時に起きる、またはステップ(a)の後に続くことを特徴とする方法。
- 請求項11に記載の方法において、前記ルシフェラーゼがセレンテラジンを基質として利用することを特徴とする方法。
- 請求項11に記載の方法において、前記ルシフェラーゼが海洋生物起源であることを特徴とする方法。
- 請求項11に記載の方法において、活性状態の前記ルシフェラーゼがシステイン残基間にジスルフィド結合を有することを特徴とする方法。
- 請求項11に記載の方法において、前記方法がレポータ遺伝子アッセイの一部であることを特徴とする方法。
- 請求項11に記載の方法が、前記サンプルを非イオン性界面活性剤と接触させるステップをさらに含むことを特徴とする方法。
- 請求項11に記載の方法が、前記サンプルを両性イオン性界面活性剤と接触させるステップをさらに含むことを特徴とする方法。
- 請求項11に記載の方法において、前記試薬組成物がキレータおよび酸化還元バッファの1つまたは2つを含むことを特徴とする方法。
- 細胞または細胞サンプル中の組み換えルシフェラーゼの量および/または活性を測定する方法において、当該方法が:
(a)前記細胞または細胞サンプルを可溶化して細胞可溶化物を調製するステップ;
(b)前記細胞可溶化物を、臭化物陰イオン、キレータ、酸化剤、酸化還元バッファ、およびpHが8.0よりも高いバッファの1つまたは2つ以上を含む試薬組成物と接触させるステップ;
(c)前記ルシフェラーゼの基質を加えるステップ;
(d)前記サンプル中の生物発光を検出するステップ;
を含み、前記組み換えルシフェラーゼは、機能的シグナルペプチドを欠き、野生型として分泌されるルシフェラーゼの非分泌型変種または誘導体であることを特徴とする方法。 - 細胞または細胞サンプル中の組み換えルシフェラーゼの量および/または活性を測定する方法において、当該方法が:
(a)前記細胞または細胞サンプルを可溶化して細胞可溶化物を調製するステップ;
(b)前記細胞可溶化物を、前記ルシフェラーゼを活性状態に転換できる環境を与える試薬組成物と接触させるステップであって、当該試薬組成物が臭化物陰イオン、キレータ、酸化剤、酸化還元バッファ、およびpHが8.0よりも高いバッファの1つまたは2つ以上を含むステップ;
(c)前記ルシフェラーゼの基質を加えるステップ;
(d)前記サンプル中の生物発光を検出するステップ;
を含み、前記組み換えルシフェラーゼは、機能的シグナルペプチドを欠き、野生型として分泌されるルシフェラーゼの非分泌型変種または誘導体であることを特徴とする方法。 - 請求項22に記載の方法において、前記ルシフェラーゼの活性状態への転換が、不活性状態から活性状態への転換、または、部分的活性または低い活性状態からより高い活性状態への転換を意味することを特徴とする方法。
- 請求項22に記載の方法において、ステップ(c)がステップ(b)と同時に起きる、またはステップ(b)の後に続くことを特徴とする方法。
- 請求項22に記載の方法において、前記試薬組成物がキレータおよび酸化還元バッファの1つまたは2つを含むことを特徴とする方法。
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