Microsoft Word _Cdc2.Cdk1 Kinase Assay_Kit_130214_..doc

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1 Printed: May 29, 1997 Revised: February 14, 2013 (Ver. 6.1) For Research Use Only. Not for use in diagnostic procedures. Non Radioisotopic Kit for Measuring Cdc2/Cdk1 Kinase Activity MESACUP Cdc2/Cdk1 Kinase Assay Kit CODE No wells MEDICAL & BIOLOGICAL LABORATORIES CO., LTD. URL TEL

2 CONTENTS 1. Intended Use Summary and Explanation Principle Materials provided Storage and Stability Materials and equipment required Precautions Procedure Additional information Related products References

3 Before use, thoroughly read all Instructions. Intended Use The MESACUP Cdc2/Cdk1 Kinase Assay Kit is a non-radioisotopic assay kit for measuring Cdc2/Cdk1 kinase activity. For Research Use Only. Not for use in diagnostic procedures. Summary and Explanation Phosphorylation and dephosphorylation of proteins, which are catalyzed by protein kinase and protein phosphatases respectively, were reported to regulate major cell functions 20)-22). Specially, Cdc2/Cdk1 kinase and its homologues play an essential role in the regulation of the cell cycle and gene transcription 15), 17), 18), 19). Therefore the measurement of kinase activity is indispensable for study of cell function. To assay for Cdc2/Cdk1 kinase activity, the protein substrate, Histone H1, and radioactive ATP have been commonly used. This assay can be expensive, time consuming and utilize large amounts of radioactive ATP. Furthermore, Histone H1 is not a specific substrate for Cdc2/Cdk1 kinase. The phosphorylation of Histone H1 is observed with several other protein kinases 16). These drawbacks involve limitation of research installation and affection of cold ATP in the assay. MBL has developed the MESACUP Cdc2/Cdk1 Kinase Assay Kit to provide a simple, reliable and non-radioactive method for measuring Cdc2/Cdk1 kinase activity. Principle The MESACUP Cdc2/Cdk1 Kinase Assay Kit is based on an enzyme linked immunosorbent assay (ELISA) that utilizes a synthetic peptide as a substrate for the Cdc2/Cdk1 kinases and a monoclonal antibody recognizing the phosphorylated form of the peptide (Fig. 1). This method is as sensitive as the radioactive one and is less affected by concentrations of ATP present in the reaction mixture. The assay can be performed on crude cell extracts, column fractions or purified enzymes. The assay kit is of value for purification of Cdc2/Cdk1 kinase. screening of inhibitors or activators of Cdc2/Cdk1 kinase. detecting pharmacological effects on Cdc2/Cdk1 kinase. -1-

4 Fig. 1 Diagram of the Cdc2/Cdk1 kinase assay (1) Cdc2/Cdk1 kinase present in the sample catalyzes phosphorylation of biotinylated MV peptide. (2) Transfer the reaction mixture to anti-phospho MV peptide monoclonal antibody (4A4) immobilized microwell. (3) The biotinylated phospho MV peptides are bound to anti-phospho MV peptide monoclonal antibody (4A4). (4) And, the peptides are subsequently detected with streptavidin conjugated to horseradish peroxidase. (5) The horseradish peroxidase substrate is then added to the microwell and the intensity of the color is measured photometrically at 492 nm. -2-

5 Materials provided Each kit contains; Name Materials Quantity (1 kit) Microwells Microwell strips coated with the monoclonal antibody 4A4 8-well strip 12 strips Reaction buffer Buffer for phosphorylation reaction (10 conc.) (250 mm HEPES buffer (ph 7.5), 100 mm MgCl 2 ) 0.5 ml 1 vial Biotinylated MV peptide MV peptide; SLYSSSPGGAYC 0.27 ml 1 vial POD conjugated streptavidin POD conjugated streptavidin (ready-to-use) 6.0 ml 2 vials Wash concentrate Buffer for washing microwells (10 conc.) 50 ml 2 bottles Substrate A o-phenylenediamine 3 tablets Substrate B H 2 O 2 50 ml 1 bottle Phosphorylation stop reagent PBS containing 50 mm EDTA (ready-to-use) 6.0 ml 2 vials Stop solution 20% H 3 PO 4 (ready-to-use) (irritant) 40 ml 1 bottle Storage and Stability All kit components must be stored at 2-8 C. All reagents are stable for two and a half years (30 months) after manufacturing when stored at the indicated conditions. Materials and equipment required Adenosine 5 -triphosphate disodium salt (ATP) Water bath at 30 C Microplate reader (492 nm) Automatic plate washer or wash bottle Adjustable micropipette Multichannel micropipette 1.5 ml of centrifuge tubes Microplate holder Disposable reagent vessels Paper towels Distilled water Sample buffer 50 mm Tris-HCl (ph 7.5), 0.5 M NaCl, 5 mm EDTA, 2 mm EGTA, 0.01% Brij-35, 1 mm PMSF, 0.05 mg/ml leupeptin, 50 mm 2-mercaptoethanol, 25 mm β-glycerophosphate, 1 mm Sodium-orthovanadate -3-

6 Precautions 1. Microwells which are not immediately required should be returned to the ziplock pouch, which must be carefully resealed to avoid moisture absorption. 2. POD conjugated streptavidin contains thimerosal (< 0.1 ppm). Protect eyes and skin and handle with care. 3. Substrate A is o-phenylenediamine (20.6%). Handle with care. 5. Substrate B contains H 2 O 2 (0.01%). 6. Stop Solution is 20% phosphoric acid. As it is corrosive product, protect eyes and skin and handle with care. 7. This kit is intended for research use only. Not for use in diagnostic procedures. Do not use internally or externally in humans or animals. Procedure (recommended) Preparation of Reagents 1. ATP (0.1 M) Dissolve 60 mg ATP in 0.8 ml of H 2 O. Adjust the ph to 7.0 with 0.1 M NaOH. Adjust the volume to 1 ml with H 2 O. Dispense the solution into small aliquots and store at -20 C. The molar absorption coefficient of ATP ε259 = (ph 7.0) Just prior to the assay, dilute 0.1 M ATP with distilled water and prepare 1 mm ATP. 2. Wash solution Prepare Wash solution by diluting 1 part of the Wash concentrate with 9 parts of distilled water. This Wash solution is stable for 1 month at 4 C. Example: Dilute 10 ml of Wash concentrate with 90 ml of distilled water. 3. Substrate solution Substrate solution must be prepared just prior to color development. Prepare Substrate solution by dissolving one tablet of Substrate A in 12 ml of Substrate B. * Keep the solution in the dark and use as soon as possible. * Use disposable or clean pipette and vessel, as Substrate solution is easily oxidized by metal ions. -4-

7 Preparation of samples Prepare according to the Additional information. Assay procedure Depends on its source, each application for Cdc2/Cdk1 kinase activity may exhibit different optimum reaction temperature and incubation time. Therefore, the most suitable condition may need to be determined for each application. Recommended incubation temperature is at 30 C for phosphorylation reaction. STEP 1: Phosphorylation reaction (This reaction makes enough volume to be split into two different wells to permit measuring the samples in duplicate. If you are not measuring each well in duplicate, you will need to divide the volumes in half or you will run short of the biotinylated peptide.) The assay procedure is summarized in the Table 1. It is advisable to set up the reactions on ice. 1) Label the sample tubes and place in a rack. 2) Pipette 5 µl of sample or Sample buffer into each appropriate tube. 3) Pipette 5 µl of 10 cdc2 Reaction buffer into each tube. 4) Pipette 5 µl of Biotinylated MV peptide into each tube. 5) Pipette 30 µl of distilled water into each tube. 6) Start the phosphorylation reaction by adding 5 µl of 1 mm ATP solution. Mix well the contents of the tube. 7) Incubate for 5-30 minutes at 30 C (use a water bath). 8) Terminate the reaction with 200 µl of Phosphorylation stop reagent to each tube. 9) Mix well the terminated reaction mixture and centrifuge for 15 seconds at 14,000 rpm. 10) Separate phosphorylated peptide as described in the following section. -5-

8 Sample (µl) Blank (µl) Sample 5 - Sample buffer cdc2 Reaction buffer 5 5 Biotinylated MV peptide 5 5 Distilled water mm ATP 5 5 Mix and incubate for 5-30 minutes at 30 C Phosphorylation stop reagent Centrifuge for 15 seconds at 14,000 rpm Detect the phosphorylated MV peptide by ELISA Table 1 Summary of the phosphorylation assay Final concentration of the reaction mixture; 25 mm HEPES buffer (ph 7.5), 10 mm MgCl 2, 0.1 mm ATP STEP 2: Detection of Cdc2/Cdk1 kinase activity with antibody Perform all assays in duplicate. 1) Transfer 100 µl of the terminated reaction mixture to each microwell strip coated with a monoclonal antibody 4A4. 2) Incubate for 60 minutes at 25 C. 3) Aspirate or discard the well contents, then fill each well with Wash solution. Aspirate or discard the Wash solution in the wells. Repeat this step another 4 times. Tap the plate on a paper towel to remove any remaining Wash solution. Be carefully not to dry the wells. 4) After completely removing any remaining wash solution, pipette 100 µl of POD conjugated streptavidin into each well. 5) Incubate for 30 minutes at 25 C. -6-

9 STEP 3: Washing Wash the wells again following the STEP 2-3) procedure. STEP 4: Substrate incubation 1) After the washing, pour Substrate solution into vessel. Add 100 µl of Substrate solution into each well. 2) Incubate for 3-5 minutes at 25 C. STEP 5: Stopping reaction Pour Stop solution (ready-to-use) into the vessel. Add 100 µl of Stop solution into each well. Reading Read the absorbance of each well at 492 nm using a plate reader. * Ensure that the back of the plate is clean and dry, and that no air bubbles are present on the surface of the liquid in the wells before reading. Fig. 2 Dilution curve of Cdc2/Cdk1 kinase A: Radio Active (RI) method, B: MESACUP Cdc2/Cdk1 Kinase Assay Kit -7-

10 Additional information Cell culture and sample preparation HeLa cells were cultured in Dulbecco s modified Eagle s medium supplemented with 10% fetal calf serum. Just before the cells reached confluence, 20 ng/ml (final concentration) colcemid was added to the culture media hours later, the mitotic cells were scraped with a rubber policeman. After 3 times washing with cold PBS, cell pellets ( cells) were suspended in 1 ml of cold Sample buffer, and were sonicated for seconds on ice. Cell extract was separated by centrifugation at 100,000 g for 1 hour at 4 C. Sample buffer: (It is also described in Materials and equipments required) 50 mm Tris-HCl (ph 7.5), 0.5 M NaCl, 5 mm EDTA, 2 mm EGTA, 0.01% Brij-35, 1 mm PMSF, 0.05 mg/ml leupeptin, 50 mm 2-mercaptoethanol, 25 mm β-glycerophosphate, 1 mm Sodium-orthovanadate Related products 5230 MESACUP Protein Kinase Assay Kit References 1) Enokido, Y., et al., J. Cell Biol. 189, (2010) 2) Boulware, M. J. and Marchant, J. S., J. Physiol. 586, (2008) 3) Kawahara, M., et al., Reproduction 130, (2005) 4) Kano, F., et al., Genes Cells 10, (2005) 5) Ito, J., et al., Reproduction 129, (2005) 6) Ito, J., et al., Reproduction 128, (2004) 7) Kishigami, S., et al., Biol. Reprod. 70, (2004) 8) Ito, J., et al., Biol. Reprod. 70, (2004) 9) Ito, J., et al., Biol. Reprod. 69, (2003) 10) Shimada, M., et al., Reproduction 124, (2002) 11) Shimada, M., et al., Biol. Reprod. 65, (2001) 12) Shimada, M., et al., Biol. Reprod. 64, (2001) 13) Shimada, M. and Terada, T. Biol. Reprod. 64, (2001) 14) Dobashi, Y., et al., J. Biol. Chem. 275, (2000) -8-

11 15) Tsujimura, K., et al., J. Biol. Chem. 269, (1994) 16) Marshak, D., et al., J. Cell Biochem. 45, (1991) 17) Less, J., et al., EMBO J. 10, (1991) 18) Peter, M., et al., Cell 61, (1990) 19) Moreno, S. and Nurse, P., Cell 61, (1990) 20) Nishizuka, Y., Nature 334, (1988) 21) Edelman, A. M., et al., Ann. Rev. Biochem. 56, (1987) 22) Montminy, M. R. and Bilezikjian, L. M., Nature 328, (1987) 23) Nishizuka, Y., Science 233, (1986) 24) Wolf, M., et al., Nature 317, (1985) Manufacturer MEDICAL & BIOLOGICAL LABORATORIES CO., LTD. URL TEL

12 はじめに MESACUP Cdc2/Cdk1 Kinase Assay Kit は ラジオアイソトープを必要としない Cdc2/Cdk1 kinase 活性測定試薬です プロテインキナーゼ プロテインフォスファターゼによるリン酸化及び脱リン酸化は cell function の調節に関与していると報告されています 20)-22) 特に Cdc2/Cdk1 kinase とそのホモログは 細胞周期の進行と関連遺伝子の転写調節に必須です 15), 17), 18), 19) 従来 Cdc2/Cdk1 kinase 活性のアッセイには 基質としてヒストン H1 を用い 放射性 ATP を利用した系が用いられてきました このアッセイ系は 放射性 ATP の使用量が多く 且つ 時間により減衰するため 費用がかかります また ヒストン H1 は Cdc2/Cdk1 kinase に特異的な基質ではなく 他の数種のプロテインキナーゼの基質にもなっています 16) 放射性 ATP を用いた方法の場合 サンプル中に含まれる ATP の影響などもありアッセイ系として限界があります MBL は ラジオアイソトープを必要とせず Cdc2/Cdk1 kinase 活性を測定可能なキット MESACUP Cdc2/Cdk1 Kinase Assay Kit を開発しました このキットは Cdc2/Cdk1 kinase の基質となる合成ペプチドとリン酸化ペプチドを特異的に認識するモノクローナル抗体を用いた ELISA 法を利用した試薬です この方法は 放射性 ATP を用いた方法と同等の感度があり 反応溶液中の ATP 濃度による影響をほとんど受けません 特徴 1) 放射性 ATP を必要としないため RI 施設の無い研究室でも Cdc2/Cdk1 kinase の活性測定ができ 廃液等の処理も通常におこなえます 2) 高濃度 ATP(2 mm) 存在下でも測定できます 3) 多数のサンプルを短時間で測定できます 4) 長期間保存することができます (2-8 C 1 年 ) -10-

13 測定原理 MESACUP Cdc2/Cdk1 Kinase Assay Kit は Cdc2/Cdk1 kinase の基質となる合成ペプチドとリン酸化ペプチドを特異的に認識するモノクローナル抗体を用いた ELISA 法を利用した試薬です 試験管内で Cdc2/Cdk1 kinase サンプルとビオチン標識 MV peptide を反応させます リン酸化されたビオチン標識 MV peptide を 抗リン酸化 MV peptide モノクローナル抗体感作マイクロカップに移して反応させます さらに ストレプトアビジン - ペルオキシダーゼを反応させた後 o-phenylenediamine(opd) と過酸化水素を添加してペルオキシダーゼにより発色させることにより 酵素反応溶液中のキナーゼの活性量を間接的に測定します Fig. 1 MESACUP Cdc2/Cdk1 Kinase Assay Kit 測定原理 -11-

14 キット構成 Name Materials Quantity (1 kit) Microwells Microwell strips coated with the monoclonal antibody 4A4 8-well strip 12 strips Reaction buffer Buffer for phosphorylation reaction (10 conc.) (250 mm HEPES buffer (ph 7.5), 100 mm MgCl 2 ) 0.5 ml 1 vial Biotinylated MV peptide MV peptide; SLYSSSPGGAYC 0.27 ml 1 vial POD conjugated streptavidin POD conjugated streptavidin (ready-to-use) 6.0 ml 2 vials Wash concentrate Buffer for washing microwells (10 conc.) 50 ml 2 bottles Substrate A o-phenylenediamine 3 tablets Substrate B H 2 O 2 50 ml 1 bottle Phosphorylation stop reagent PBS containing 50 mm EDTA (ready-to-use) 6.0 ml 2 vials Stop solution 20% H 3 PO 4 (ready-to-use) (irritant) 40 ml 1 bottle 保存 2-8 C にて保存してください 有効期間 製造後 2 年 6 ヶ月 (30 ヶ月 ) 準備するもの Adenosine 5 -triphosphate disodium salt (ATP) ウォーターバス (30 C) マイクロプレートリーダー (492 nm) プレートウォッシャーまたは洗浄ビン マイクロピペット マルチチャンネルマイクロピペット 1.5 ml マイクロチューブ マイクロカップ専用ホルダー リザーバー ペーパータオル 精製水 Sample buffer 50 mm Tris-HCl (ph 7.5), 0.5 M NaCl, 5 mm EDTA, 2 mm EGTA, 0.01% Brij-35, 1 mm PMSF, 0.05 mg/ml leupeptin, 50 mm 2-mercaptoethanol, 25 mm β-glycerophosphate, 1 mm Sodium-orthovanadate -12-

15 操作上の留意点 1. 抗体感作マイクロカップは湿気をきらいますので 十分室温 (20-25 C) に戻してから開封して下さい また 開封後はアルミ袋のチャックを確実に締めて保存して下さい 2. 本試薬の構成品のうち POD conjugated streptavidin にはチメロサール (0.1 ppm 未満 ) が含まれます 使用の際には十分注意して取り扱って下さい 3. Substrate A は o-phenylenediamine(20.6%) です 使用の際には十分注意して取り扱って下さい 4. Substrate B には 0.01% 過酸化水素 (H 2 O 2 ) が含まれます 濃度は 0.01% ですので毒物には該当しませんが 誤って目や口に入ったり 皮膚に付着した場合は水で十分に洗い流すなどの応急処置を行い 必要があれば 医師の手当てを受けて下さい 5. 本試薬の構成品のうち Stop solution には 20% リン酸 (H 3 PO 4 ) を用いています 使用の際には十分注意して取り扱って下さい 6. 本試薬は研究用試薬です ヒトの体内に用たり 診断の目的に使用しないで下さい 操作法 試薬の調製 1. ATP(0.1 M) ATP60 mg を 0.8 ml の精製水に溶解し 0.1 M NaOH で ph 7.0 に調整した後 精製水で 1 ml にします 0.1 M ATP は 凍結融解の繰り返しを避けるため 小分けして -20 C で凍結保存してください モル分子吸収係数 ATP ε259 = (ph 7.0) 使用直前に 精製水で 1 mm に調整してください 洗浄液 ] 2. Wash solution [ 洗浄液 Wash Concentrate を精製水で 10 倍希釈し Wash solution とします Wash solution は 4 C で保存すると およそ 1 ヶ月間安定です 例 : 10 ml の Wash concentrate に 90 ml の精製水を加え 10 倍希釈します 酵素基質溶液 ] Substrate solution は保存できないので 発色直前に調製します 室温に戻した 12 ml の Substrate B に 1 錠の Substrate A を加えて溶解し Substrate solution とします 3. Substrate solution [ 酵素基質溶液 * Substrate solution はできる限り遮光してください * Substrate solution は金属イオンにより酸化されやすいので取り扱いは ディスポーザブ ル あるいは精製水で十分にすすいだきれいな器具を使用して下さい -13-

16 サンプルの調製 後述の Additional information を参照してください 測定の手順 アッセイの準備は氷中でおこなってください Cdc2/Cdk1 kinase 活性測定では 各々のアプリケーションで最適な反応温度やインキュベーション時間が異なります よって 各々のアプリケーションにおける最適反応温度 及びインキュベーション時間を設定する必要があります リン酸化反応における温度は 30 C をお勧めします STEP 1. < リン酸化反応 > Table 1 に測定手順概要を示します ( 構成試薬の容量は 二重測定をおこなった場合に十分な容量に設定してあります 二重 測定をおこなわない場合には Table 1 の方法に従って測定サンプルを調製する時に サン プル及び試薬量を減量してください ) 1) サンプルにラベルし ラックに入れます 2) 適当な試験管にサンプルあるいは Sample buffer 5 µl を入れます 3) 各試験管に 10 cdc2 Reaction buffer 5 µl を加えます 4) 各試験管に Biotinylated MV peptide 5 µl を加えます 5) 各試験管に精製水 30 µl を加えます 6) 各試験管に 1 mm ATP solution 5 µl を加え よく混合します 1 mm ATP solution を加えた時点より反応が開始します 7) 30 C で 5-30 分間インキュベートします ( ウォーターバスを使用します 実験の目的により反応時間を変更してください ) 8) Phosphorylation stop reagent 200 µl を加えてリン酸化反応を停止させます 9) 反応を停止したリン酸化ペプチド溶液を混合し 14,000 rpm で 15 秒間遠心します -14-

17 Table 1 リン Sample (µl) Blank (µl) Sample 5 - Sample buffer cdc2 Reaction buffer 5 5 Biotinylated MV peptide 5 5 Distilled water mm ATP 5 5 Mix and incubate for 5-30 minutes at 30 C Phosphorylation stop reagent Centrifuge for 15 seconds at 14,000 rpm Detect the phosphorylated MV peptide by ELISA リン酸化反応測定手順概要 リン酸化酸化ペプチドペプチド溶液溶液の最終濃度 ; 25 mm HEPES buffer(ph 7.5), 10 mm MgCl 2, 0.1 mm ATP STEP 2. < 抗体反応 > 二重測定を推奨推奨しますします 1) STEP 1. で反応させたリン酸化ペプチド溶液をマイクロカップ 1 ウェルにつき 100 µl 添加します 2) 25 C で 60 分間反応させます 3) ウェル内の反応液を完全に除去後 ウェルを十分な量の Wash solution で満たし 捨てます これをさらに 4 回行います (5 回洗浄 ) その後 プレートをペーパータオルなどに軽くたたきつけて完全に洗浄液を除去してください また 洗浄中にウェルを乾燥させないでください 4) POD conjugated streptavidin を 1 ウェルにつき 100 µl 添加します 5) 25 C で 30 分間反応させます STEP 3. < 洗浄 > STEP 2. -3) と同様にマイクロカップを洗浄します -15-

18 STEP 4. < 酵素反応 > 1) Substrate solution を 1 ウェルにつき 100 µl 添加します 2) 25 C で 3-5 分間反応させます STEP 5. < 反応停止 > リザーバーに移した Stop solution をマルチチャンネルマイクロピペットで 100 µl ずつ各ウェルに添加し 反応を停止させます 吸光度の測定 マイクロプレートリーダーにマイクロカップをセットして 波長 492 nm の吸光度を測定します * プレートの裏側は汚れのない乾いた状態を保つようにしてください また 吸光度を測定 する前にウェル内に気泡がないことを確認してください Fig. 2 Dilution curve of Cdc2/Cdk1 kinase A: Radio Active (RI) method, B: MESACUP Cdc2/Cdk1 Kinase Assay Kit -16-

19 Additional information Cell culture and sample preparation HeLa cells を 10% fetal calf serum を添加した Dulbecco s modified Eagle s medium で培養します 細胞が confluence になる直前に colcemid を培養液に添加します ( 最終濃度 20 ng/ml) 時間培養後 分裂期細胞を集めます 細胞を冷却した PBS にて洗浄後 cell pellets( cells) を 1 ml の冷却した Sample buffer に加え 氷中で 秒ソニケートします 4 C 100,000 g で 1 時間遠心します Sample buffer:( 準備するものするもので紹介したものと同じです ) 準備するもの 50 mm Tris-HCl (ph 7.5), 0.5 M NaCl, 5 mm EDTA, 2 mm EGTA, 0.01% Brij-35, 1 mm PMSF, 0.05 mg/ml leupeptin, 50 mm 2-mercaptoethanol, 25 mm β-glycerophosphate, 1 mm Sodium-orthovanadate 関連製品 5230 MESACUP Protein Kinase Assay Kit 参考文献 1) Enokido, Y., et al., J. Cell Biol. 189, (2010) 2) Boulware, M. J. and Marchant, J., S. J. Physiol. 586, (2008) 3) Kawahara, M., et al., Reproduction 130, (2005) 4) Kano, F., et al., Genes Cells 10, (2005) 5) Ito, J., et al., Reproduction 129, (2005) 6) Ito, J., et al., Reproduction 128, (2004) 7) Kishigami, S., et al., Biol. Reprod. 70, (2004) 8) Ito, J., et al., Biol. Reprod. 70, (2004) 9) Ito, J., et al., Biol. Reprod. 69, (2003) 10) Shimada, M., et al., Reproduction 124, (2002) 11) Shimada, M., et al., Biol. Reprod. 65, (2001) 12) Shimada, M., et al., Biol. Reprod. 64, (2001) 13) Shimada, M. and Terada, T., Biol. Reprod. 64, (2001) 14) Dobashi, Y., et al., J. Biol. Chem. 275, (2000) 15) Tsujimura, K., et al., J. Biol. Chem. 269, (1994) 16) Marshak, D., et al., J. Cell Biochem. 45, (1991) 17) Less, J., et al., EMBO J. 10, (1991) -17-

20 18) Peter, M., et al., Cell 61, (1990) 19) Moreno, S. and Nurse, P., Cell 61, (1990) 20) Nishizuka, Y., Nature 334, (1988) 21) Edelman, A. M., et al., Ann. Rev. Biochem. 56, (1987) 22) Montminy, M. R. and Bilezikjian, L. M., Nature 328, (1987) 23) Nishizuka, Y., Science 233, (1986) 24) Wolf, M., et al., Nature 317, (1985) 製造元 株式会社医学生物学研究所 URL TEL

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