JP4948530B2 - ホルマリン固定組織からのタンパク質の定量的測定 - Google Patents
ホルマリン固定組織からのタンパク質の定量的測定 Download PDFInfo
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N1/31—Apparatus therefor
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Toxicology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
(b)切片がパラフィンを除去される;
(c)試験すべき組織範囲が、組織切片から手でまたはレーザー顕微解剖によって切り出される(随意的に組織全体が試験されうる);
(d)切り出された組織片が、界面活性剤を含みおよびタンパク質分解活性を有する化合物を含まない緩衝液へ移される。タンパク質の完全性を確実にするため、たとえばトリプシンまたはプロテイナーゼKといったプロテアーゼは使用してはならない;
(e)緩衝液中の試料はまず煮沸される(95℃から100℃へ)。インキュベート時間は、たとえば5分間ないし40分間と変化しうる。煮沸の時間は、たとえば、試料のサイズに依存しうる;
(f)試料は次いで60℃を上回る温度(たとえば80℃)にてインキュベートされる。60℃を上回るインキュベート時間は、たとえば1時間ないし6時間と変化しうる。60℃より高温での最大インキュベート時間は、しかし、16時間未満とすべきである;
(g)インタクトタンパク質はここで十分な量で溶液中に存在し、および、たとえば定量されうる。
(a)タンパク質分解活性を有する化合物を含まない緩衝液系、および
(b)界面活性剤
を含む、ホルマリン固定生物試料からのインタクトタンパク質の定量的抽出のためのキットを提供する。
下記で、本方法に記載の典型的なタンパク質抽出が詳細に記載される。標準的方法と比較したこの方法の顕著な改善(高いタンパク質収量)が図2に図示される。実験者は、手順のうちどれかの段階をいくらか改変しうる。いくらか異なる組成およびpH値を有する緩衝液が使用されうる。たとえばSDSといった界面活性剤の使用、95℃から100℃での試料の煮沸、および以降の60℃より高温(たとえば80℃)でのインキュベートは、しかし、極めて重要である;さらに、プロテアーゼを使用してはならない。各熱処理の時間および緩衝液の量は変化しうる。同じく変化しうるのは、組織の手動破砕である。破砕は機械的に(たとえば乳棒を用いて)または、たとえば、超音波を用いて実施されうる。典型的な時間および量をここに列記する。ホルマリン固定組織由来の2種類のタンパク質の相関の結果を図3に示す。ホルマリン固定組織由来のインタクトタンパク質を用いた最初のタンパク質アレイを図4に示す(逆相タンパク質アレイ、ドットブロット)。
1.同一のパラフィンブロックから2x10μm切片を作製する
2.切片を脱パラフィンする
2.1.10分間キシレン、2回繰り返し
2.2.10分間100%エタノール
2.3.10分間96%エタノール
2.4.10分間70%エタノール
2.5.切片を蒸留水へ移す
3.切片を蒸留水から取り出し、短時間乾燥させる(しかし完全に乾燥させない)
4.組織範囲をカニューレで掻き取る
5.カニューレの切り取った組織を抽出緩衝液(EB)* 100μlへ移す(1.5ml反応容器当たり2切片からの組織)
6.抽出緩衝液中でテフロン乳棒を用いてよく磨砕し、氷上に保持する
7.ボルテックスにかけ、氷上に保持する
8.段階6から7を1回繰り返し
9.溶液をシリンジを通じて数回注意深く吸引する
10.ボルテックスにかけ、氷上に保持する
11.ボルテックスにかけ、氷上に保持する(泡が多ければ:短時間遠心分離)
12.20分間100℃(水浴)
13.2時間80℃(振とう機、750rpm)
14.15分間遠心分離、4℃、12500rpm
15.上清を新しい1.5ml反応容器へ→準備完了したタンパク質溶解物
a.5xレムリ緩衝液ストック(ブロモフェノールブルーを含まない)10mlバッチ
2.5 ml 1.25Mトリス/HCl(pH6.8)
4.5 ml グリセロール
2.8 ml ベータ−メルカプトエタノール
1 g SDS
蒸留水で10mlとする
[1Mトリスは121.14gに相当;1.25Mは151.43gに相当
1000ml−−151.43g;50ml−−7.6g;濃HClでpH6.8に調整]
2.5 ml T−PER(登録商標)(ピアース社(Pierce))
+ 2.5 ml 2xレムリ(Laemmli)緩衝液
+1/2 コンプリートミニプロテアーゼインヒビター錠(バイエル社(Bayer))
本発明に記載の方法の別の一実施形態では、DTT(1,4−ジチオ−DL−スレイトール)が用いられる。DTTは好ましくは、単離されたタンパク質の、たとえばバイオラッド社(BioRad)のDCプロテインアッセイキット(登録商標)またはピアース社(Pierce)のマイクロBCAアッセイキット(登録商標)を用いた定量に用いられる(図5)
2.切片を脱パラフィンする
2.1.10分間キシレン、2回繰り返し
2.2.10分間100%エタノール
2.3.10分間96%エタノール
2.4.10分間70%エタノール
3.抽出緩衝液(EB2)*100μlを加える
4.ボルテックスにかけ、氷上に保持する
5.溶液をピペットを通じて数回注意深く吸引する
6.ボルテックスにかけ、氷上に保持する(泡が多ければ:短時間遠心分離)
7.20分間100℃(水浴)
8.2時間80℃(振とう機、750rpm)
9.15分間遠心分離、4℃、12500rpm
10.上清を新しい1.5ml反応容器へ移す→準備完了したタンパク質溶解物
11.市販のタンパク質定量キットによって定量
90mMトリス/HCl(pH6.8)
20%グリセロール
2%SDS
1mM DTT(1,4−ジチオ−DL−スレイトール)
Claims (20)
- 生物試料が緩衝液中でタンパク質を放出するのに適した温度にてインキュベートされる、ホルマリン固定生物試料からのタンパク質の抽出のための方法であって、前記緩衝液が界面活性剤を含み、かつタンパク質分解活性を有する化合物を含まず、および緩衝液中の生物試料が5から40分間まず煮沸され、および次いで60℃より高く、かつ95℃より低い温度にて20分間から16時間の期間さらにインキュベートされる点で特徴づけられる、方法。
- 界面活性剤がSDS、デオキシコール酸ナトリウム、CHAPS、トリトン(Triton)X100(登録商標)、ノニデット(Nonidet)P40(登録商標)またはツイーン(Tween)20(登録商標)である、請求項1に記載の方法。
- 緩衝液がさらに還元剤を含む、請求項1又は2に記載の方法。
- 還元剤が、1,4−ジチオ−DL−スレイトール、DTE、TCEPまたはMEAである、請求項3に記載の方法。
- 生物試料がパラフィンに包埋されたホルマリン固定試料である場合、生物試料がタンパク質抽出前に脱パラフィンされる、請求項1から4のいずれかに記載の方法。
- 抽出後にタンパク質がさらに分画される、請求項1から5のいずれかに記載の方法。
- 抽出されたタンパク質が一またはいくつかの方法段階を用いて分画される、請求項6に記載の方法。
- 抽出されたタンパク質が後で定量される、請求項1から7のいずれかに記載の方法。
- タンパク質の定量がローリー(Lowry)法またはBCAによって実施される、請求項8に記載の方法。
- 抽出されたタンパク質が、タンパク質分解酵素、グリコシダーゼ、またはホスファターゼで処理される、請求項1から9のいずれかに記載の方法。
- タンパク質分解酵素がトリプシン、キモトリプシン、プロテイナーゼK、パパイン、ペプシン、プロナーゼ、エンドプロテイナーゼLys−C、又はエンドプロテイナーゼglu−Cである、請求項10に記載の方法。
- グリコシダーゼがエンドグリコシダーゼH、N−グリコシダーゼF、又はノイラミニダーゼである、請求項10又は11に記載の方法。
- タンパク質が少なくとも一つの生化学検定法に用いられる、請求項1から12のいずれかに記載の方法。
- 生化学検定法がタンパク質アレイである、請求項13に記載の方法。
- タンパク質アレイがマイクロアレイである、請求項14に記載の方法。
- タンパク質アレイがサンドイッチイムノアレイ、抗原捕捉アレイ、又は直接タンパク質アレイである、請求項14に記載の方法。
- 生化学検定法が、一つ以上の診断上または臨床上関連するマーカータンパク質の測定に役立つ、請求項13から16のいずれか1項に記載の方法。
- 少なくとも2つの生物試料に由来するマーカータンパク質が互いに比較される、請求項17に記載の方法。
- 生物試料が組織試料である、請求項1から18のいずれかに記載の方法。
- タンパク質分解活性を有する化合物がプロテアーゼである、請求項1から19のいずれかに記載の方法。
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DE102005023011A DE102005023011A1 (de) | 2005-05-19 | 2005-05-19 | Quantitative Proteinbestimmung von Formalin fixiertem Gewebe |
DE102005023011.3 | 2005-05-19 | ||
PCT/EP2006/062198 WO2006122898A1 (de) | 2005-05-19 | 2006-05-10 | Proteinextraktion aus formalin fixiertem gewebe |
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KR (1) | KR101275335B1 (ja) |
CN (1) | CN101180310B (ja) |
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CA (1) | CA2607414C (ja) |
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US20090069549A1 (en) * | 2007-09-12 | 2009-03-12 | Elias Georges | Protein Extraction buffer, a kit comprising it and method of its use |
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WO2010065775A2 (en) | 2008-12-03 | 2010-06-10 | The United States Government As Represented By The Department Of Veterans Affairs | Pressure-assisted molecular recovery (pamr) of biomolecules, pressure-assisted antigen retrieval (paar), and pressure-assisted tissue histology (path) |
IT1392074B1 (it) * | 2008-12-04 | 2012-02-09 | Porto Conte Ricerche S R L | Procedimento per l'estrazione di proteine da campioni di tessuto biologico ffpe |
EP2193831A1 (de) * | 2008-12-05 | 2010-06-09 | Qiagen GmbH | Parallel-Extraktion von unterschiedlichen Biomolekülen aus Formalin-fixiertem Gewebe |
CN102485979B (zh) * | 2010-12-02 | 2014-02-19 | 深圳华大基因科技服务有限公司 | Ffpe样品核酸文库,其构建方法和ffpe样品分析方法 |
US9080167B2 (en) * | 2012-11-16 | 2015-07-14 | Covaris, Inc. | System and method for processing paraffin embedded samples |
US20170168055A1 (en) * | 2015-12-11 | 2017-06-15 | Expression Pathology, Inc. | SRM/MRM Assays |
WO2017136198A1 (en) * | 2016-02-01 | 2017-08-10 | Mayo Foundation For Medical Education And Research | Methods and materials for extracting chromatin |
SG11201908865WA (en) * | 2017-06-29 | 2019-10-30 | Quanticision Diagnostics Inc | Apparatus and method for absolute quantification of biomarkers for solid tumor diagnosis |
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US20090124510A1 (en) | 2009-05-14 |
WO2006122898A1 (de) | 2006-11-23 |
CN101180310A (zh) | 2008-05-14 |
DE102005023011A1 (de) | 2006-11-23 |
US8828709B2 (en) | 2014-09-09 |
KR101275335B1 (ko) | 2013-06-14 |
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