PTM PTM Cell Signaling Technology CST 1,000 CST CST p.13 CST CST 2

Transcription

1 A Guide to successful Western Blotting from Cell Signaling Technology WB

2 PTM PTM Cell Signaling Technology CST 1,000 CST CST p.13 CST CST 2

3 1 tissue or cells homogenous solution sample Preparation Lysis buffer & sonication = better release of proteins CST CST sds-page Run with appropriate controls Wet transfer To nitrocellulose membranes Blocking 5% milk in TBST Primary Antibody incubation At 4 C overnight in 5% BSA or nonfat milk* in TBST * as recommended on product datasheet detection Mix reagents immediately before use for chemiluminescent detection Washing 3 x 5 min in TBST secondary Antibody incubation For 1 hr at room temperature in 5% nonfat milk in TBST Washing 3 x 5 min in TBST imaging Chemiluminescent imaging offers greater flexibility and ease of use

4 CST sirna 4

5 適切なコントロールの組み入れが重要 U0126 TPA Jurkat Phospho-p44/42 MAPK Erk1/2 Thr2/Tyr4 Total GAPDH H3 A MEK1/2 / #4370 CST #4370 Phospho-p44/42 MAPK Erk1/2 Thr2/Tyr4 D E XP Rabbit mab CST #9411 Phospho-Tyrosine Mouse mab P-Tyr- 1 Phospho-p44/42 MAPK (Erk1/2) (Thr2/Tyr4) (D E) XP Rabbit mab #4370 TPA UO126 VEGFR c-kit FLT3 Tie2 Src CSFR EGFR PDGFR HER2 Met c-abl IR 1 TPA UO126 PDGFR Alternative Provider 1 c-kit FLT3 Tie2 Src CSFR EGFR VEGFR Met c-abl HER2 IR Jurkat Lysate recombinant proteins Jurkat Lysate recombinant proteins Alternative Provider 2 1 CST Phospho-Tyrosine # TPA UO126 EGFR PDGFR CSFR VEGFR c-kit FLT3 Tie2 Src HER2 Met c-abl IR TPA UO126 CSFR EGFR PDGFR Tie2 Src FLT3 VEGFR c-kit Met c-abl HER2 IR Jurkat Lysate recombinant proteins Jurkat Lysate recombinant proteins 5

6 CST CST 6

7 完全な溶解の徹底と新鮮な試料の使用が重要 A CST H3 B CST DNA 35% % 3 DNA RIPA C 適正な試料と分子量マーカーの組み入れが必要 SDS-PAGE CST - - g - (A) final inhibitor target concentration Aprotinin Trypsin, Chymotrypsin, Plasmin 2 µg/ml Leupeptin Lysosomal 1 µg /ml Pepstatin A Asp proteases 1 µg /ml PMSF Asp proteases 1 mm EDTA Mg & Mn metalloproteases 1 5 mm EGTA Ca metalloproteases 1 mm Na Fluoride Ser & Thr phosphatases 5 mm Orthovanadate Tyr phosphatases 1 mm Pyrophosphate Ser & Thr phosphatases 1 2 mm β-glycerophosphate Ser & Thr phosphatases 1 2 mm B CKR/ PAEC Phospho- Histone H3 (Ser Antibody #9701 C 3 1 Trk pan C17F1 Rabbit mab # Sonicated old new sorbitol Unsonicated Trk (pan) Phospho- Histone H3 (Ser) 標的タンパク質の濃度が検出レベル以下のため シグナルが検出されない 7

8 ウェット式転写最適な転写方式 % 70V m セミドライ式転写ウェット式に代わって汎用される転写方式 CST BioRad Trans-blot Turbo A A Pan-Actin (D18C11) Rabbit mab #8456 HeLa COS-7 NIH/3T3 1 Wet Transfer HeLa COS-7 NIH/3T3 HeLa COS-7 NIH/3T3 Semi-dry Transfer HeLa COS-7 NIH/3T3 HeLa COS-7 NIH/3T3 ( ) Trans-Blot Turbo ( ) 7.5 µl 4 µl 7.5 µl 4 µl Pan- Actin ゲルの厚さ 電気泳動転写の隠れた変数 CST B 1 mm 1.5 mm 1 mm 1.5 mm B Wet Transfer 1 mm gel 1.5 mm gel Semi-Dry Transfer 1 mm gel 1.5 mm gel Talin-1 C45F1 Rabbit mab #21 C6 SDS-PAGE Bio-Rad M ini-protean 1 mm Life Technologies Novex 1.5 mm Trans-Blot Turbo 1 Talin µl ゲルへのライセートの装填不足から 得られるシグナルが低い CST - g SDS-PAGE g 8

9 ドライ式転写感度の限られた転写方式 Life Technologies iblot 7 A, B CST A B iblot CST 注意 : Wet Transfer Dry Transfer HeLa NIH/3T3 HeLa NIH/3T3 HeLa NIH/3T3 HeLa NIH/3T3 HeLa NIH/3T3 HeLa NIH/3T3 1 Phosphoand total S6 Ribosomal Protein Phospho-S6 Ribosomal Protein (Ser235/236) Antibody #2211 S6 Ribosomal Protein (5G) Rabbit mab #2217 Alternate Provider Phospho-S6 (Ser235) Antibody Phospho-S6 Ribosomal Protein (Ser235/236) Antibody #2211 S6 Ribosomal Protein (5G) Rabbit mab #2217 Alternate Provider Phospho-S6 (Ser235) Antibody insulin serum A HeLa NIH/3T3 Phospho-S6 Ribosomal Protein Ser235/236 Antibody #2211 S6 Ribosomal Protein 5G Rabbit mab #2217 Phospho-S6 Ser235 iblot B Human Interferon- 1 hifn- 1 #8927 HeLa Phospho-Stat3 Tyr705 D3A7 XP Rabbit mab #9145 Phospho-Stat3 iblot 1 HeLa Wet Transfer Dry Transfer HeLa HeLa HeLa Phospho- Stat3 (Tyr705) + CST # Alternate Provider CST #9145 Alternate Provider hifn-α1 転写が不完全もしくは過剰である 0.45 m CST 0.2 m 1.5 % 5% 3 9

10 ブロッキング剤には 5% 脱脂粉乳が有効 CST TBS 5 Tween TBST 1 TBST 5 1 Milk BSA Phospho- Akt (Ser473) 希釈バッファー抗体の特異性に応じた選択が重要 5% BSA 5% TBST CST CST A C2C12 BSA Phospho-Akt Ser473 5 µl 5 µl A BSA Phospho-Akt Ser473 Antibody #9271 C2C12 TBST 5% 5% BSA インキュベーション オーバーナイトインキュベーションで抗体結合能が向上 CST 4 B, C 4 2 Phospho-Akt Ser473 PKC 4 迅速インキュベーションシステム インキュベーション時間を短縮するとシグナルも低下 CST 1 4 CST SNAPi.d. CST C C CST Phospho-p38 MAPK Thr1/ Tyr182 3D7 Rabbit mab #9215 UV NIH/3T3 C6 CST SNAPi.d 1 CST protocol NIH/3T3 C6 SNAPi.d system NIH/3T3 C6 Phosphop38 MAPK (Thr1/Tyr182) hr, 4 C 2 hr, RT Phospho- Akt (Ser473) PKCδ LY higf-i B 4 2 LY2902 #9901 Human Insulin-like Growth Factor I higf-i #8917 HeLa Phospho-Akt Ser473 Antibody #9271 PKC Antibody # UV anisomycin

11 シグナルを強くするなら TBST バッファーが最適 CST TBST A TBST Tween PBST TBST PBST 3 5 より強力なブロッキング剤での希釈によりバックグラウンドが低下 S/N CST 5% TBST B 5% 5% BSA BSA IgG MW NIH/3T3 NIH/3T3+UV Phospho-c-Jun (Ser63) MW C2C12 HeLa c-jun MW C2C12 HeLa MW Ribosomal Protein S3 HeLa COS-7 MW C2C12 C2C12+insulin Calnexin Phospho- 4E-BP1 (Thr37/46) TBST (as recommended in CST protocol) 5 sec, 4-% gel PBST (altered protocol) 5 sec, 4-% gel A TBST PBST Phospho-c-Jun Ser63 II Antibody #9261 c-jun A8 Rabbit mab #9165 Ri bosomal Protein S3 Antibody #2579 Calnexin C5C9 Rabbit mab #2679 Phospho-4E-BP1 Thr37/46 236B4 Rabbit mab #2855 TBST PBST B Phospho-Stat3 Tyr705 Antibody #9131 Human Interferon- 1 hifn- 1 #8927 Jurkat 5% 5% BSA TBST Antirabbit IgG, HRP-linked Antibody # Milk BSA Phospho- Stat3 (Tyr705) + + hifn-α1 二次抗体が細胞抽出物中の様々なタンパク質と非特異的に結合し バックグラウンドが高くなってしまう 11

12 化学発光 使いやすさの提供 SignalFire LumiGLO A 1 Akt (pan) 蛍光デジタルデータの保管と容易な定量化が可能 A Akt pan C67E7 Rabbit mab #4691 Jurkat Sig nalfire ECL Reagent #6883 LumiGLO Reagent #7003 µg CST Tween 5% TBS Phospho-p44/42 MAPK (Thr2/Tyr4) p44/42 MAPK Overlay B p44/42 MAPK U0126 TPA B U0126 #9903 M 1 TPA 12-O-Tetradecanoylphorbol-13-Acetate #4174 nm COS-7 Phospho-p44/42 MAPK Erk1/2 Thr2/Tyr4 D E XP Rabbit mab #4370 p44/42 MAPK Erk1/2 3A7 Mouse mab #

13 5% w/v BSA 1 TBS 0.1% Tween 4 注意 : A. 溶液および試薬 注意 : RODI 1. PBS:(#98) 1 PBS 1 L PBS ml 9 ml 2. TBS:(#12498) 1 TBS 1 L TBS ml 900 ml 3. 1 SDS サンプルバッファー :Blue Loading Pack (#7722) Red Loading Pack (#7723) 3 SDS 1/ DTT トリス - グリシン SDS 泳動バッファー :(#) 1 1 L ml 900 ml 5. トリス - グリシン転写バッファー :(#12539) 1 1 L ml ml 700 ml 6. TBS with Tween (TBST-X):(#9997) 1 TBST 1 L TBST ml 900 ml 7. 脱脂粉乳 :(#9999) 8. ブロッキングバッファー :5% w/v 1 TBST; 1 ml 7.5 g 1 TBST 1 ml 9. 洗浄バッファー :(#9997) 1 TBST. ウシ血清アルブミン (BSA) 11. 一次抗体希釈バッファー :5% w/v BSA 1 TBST ( ); ml BSA 1.0 g 1 TBST ml 12. ビオチン化プロテインラダー :(#7727) 13. プレステイン分子量マーカー :(#77) 14. 転写膜 :(#12369) 0.2 µm 15. HRP 標識二次抗体 :anti-rabbit (#7074); anti-mouse (#7076); anti-rat (#7077) 16. 検出試薬 :LumiGLO (#7003) SignalFire ECL Reagent (#6883) B. タンパク質のブロッティング PBS 3. 1 SDS (6 µl/ cm 0 µl/ ) 4. DNA µl SDS-PAGE ( cm cm) µl 注意 : (#77 µl/lane) (#7727 µl/lane) 8. (#12369) I. 転写膜のブロッキング 1. オプション : TBS 25 ml ml 1 3. TBST 15 ml 5 3 II. 一次抗体反応 非標識一次抗体の場合 1. ml 4 2. TBST 15 ml HRP (#7074 #7076 #7077) (1:2,000) HRP (#7075) (1:1,000-1:3,000) ml1 4. TBST 15 ml D. HRP 標識一次抗体の場合 1. ml 4 2. TBST 15 ml HRP (#7075) (1:1,000-1:3,000) ml1 4. TBST 15 ml D. ビオチン標識一次抗体の場合 1. ml 4 2. TBST 15 ml HRP (#3999) ml1 4. TBST 15 ml D. ビオチン化プロテインラダー検出用 HRP 標識ビオチン抗体は加えないでください HRP D. タンパク質の検出 1. LumiGLO (#7003; LumiGLO 0.5 ml Peroxide 0.5 ml 9.0 ml) SignalFire (#6883; 2 Reagent A 5 ml 2 Reagent B 5 ml) ml1 2. ( ) X 注意 : 2 C. 転写膜のブロッキングと抗体反応 注意 : cm cm ( cm 2 ) CST 注意 : 13

14 CST CST CST CST 14

15 CST CST ( ) #S Tris-Glycine SDS Running Buffer ( X) 0 ml 11,000 #12539S Tris-Glycine Transfer Buffer ( X) 1 L 11,000 #12498S Tris Buffered Saline (TBS- X) 1 L,000 #9997S Tris Buffered Saline with Tween (TBST- X) 0 ml 11,000 #9999S Nonfat Dry Milk 2 g 11,000 CST ( ) #7722S Blue Loading Buffer Pack 1 Pack 3, - #7723S Red Loading Buffer Pack 1 Pack 3, - * #93S Cell Lysis Buffer ( X) 15 ml 11,000 - * #96S RIPA Buffer ( X) 15 ml 11,000 - * #9852S Chaps Cell Extract Buffer ( X) 5 ml 11,000 - CST ( ) #5872S Protease/Phosphatase Inhibitor Cocktail ( X) 1 ml 48,000 4 #5871S Protease Inhibitor Cocktail ( X) 1 ml 21,000 4 #5870S Phosphatase Inhibitor Cocktail ( X) 1 ml 42,000 4 #8553S PMSF mg 4,0 CST ( ) #7727S Biotinylated Protein Ladder Detection #7727L Pack 65 mini-blots 325 mini-blots 11,000 43,000 #77S Prestained Protein Marker, Broad µl 18,000 #77L Range (Premixed Format) µl 5 72, CST ( ) #12369S Nitrocellulose Sandwiches Pack 23,000 CST ( ) #7076S Anti-mouse IgG, HRP-linked Antibody 1 ml 27,000 - #7074S Anti-rabbit IgG, HRP-linked Antibody 1 ml 27,000 - #7077S Anti-rat IgG, HRP-linked Antibody 1 ml 27,000 - #7075S Anti-biotin, HRP-linked Antibody 1 ml 27,000 - #7056S Anti-mouse IgG, AP-linked Antibody 1 ml 27,000 - #7054S Anti-rabbit IgG, AP-linked Antibody 1 ml 27,000 - #7055S Anti-biotin, AP-linked Antibody 1 ml 27,000 - #3678S #5127S #3677S #5470S #5257S #5366S #5151S Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mab Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mab (HRP Conjugate) Mouse Anti-rabbit IgG (Light-Chain Specific) (L57A3) mab Anti-mouse IgG (H+L) (DyLight 6 Conjugate) Anti-mouse IgG (H+L) (DyLight 0 Conjugate) Anti-rabbit IgG (H+L) (DyLight 6 Conjugate) Anti-rabbit IgG (H+L) (DyLight 0 Conjugate) µl 29,000 - µl 34,000 - µl 29,000-0 µl 32, µl 32, µl 32, µl 32,000 4 #3999S Streptavidin-HRP 1 ml 27,000 - CST ( ) #6883S SignalFire ECL Reagent 0 ml 35,000 #126S SignalFire Plus ECL Reagent ml 56,000 #12757S SignalFire Elite ECL Reagent ml,000 4 #7003S X LumiGLO Reagent and X Peroxide * CST 1 mm PMSF 1 Pack 32,000 4 他社からお求めいただける試薬と機器 プレキャストゲル CST Life Technologies Novex - (1.5 mm 15 ) Novex - 装置 1. : Life Technologies Novex by XCell SureLock 2. : GE Healthcare TE 22 Mini Tank Transfer Unit (Cat# ) フォームスポンジ 1. GE Healthcare (Cat# TE25F-1/8) フィルム 1. GE Healthcare Amersham Hyperfilm ECL (Cat# ) 2. Eastman Kodak BioMax Light Film (Cat# ) CST 15

16 Cell Signaling Technology CST 14 Cell Signaling Technology, Inc. SignalFire, SignalSilence, XP, CST and Cell Signaling Technology are trademarks of Cell Signaling Technology, Inc. Phototope is a registered trademark of Cell Signaling Technology and New England Biolabs. BioMax is a registered trademark of Eastman Kodak Company. Whatman is a registered trademark of Whatman International, LLC. Bio-Rad, Trans-blot, Turbo, Mini-PROTEAN, and Mini Trans-blot are trademarks of Bio-Rad Laboratories. Novex, iblot, and XCell SureLock are trademarks of Life Technologies Corporation. DyLight is a registered trademark of Thermo Fisher Scientifi c, Inc and its subsidiaries. LumiGLO is a registered trademark of Kirkegaard & Perry Laboratories. SNAPi.d. is a trademark of Millipore. Amersham and Hyperfi lm are trademarks of General Electric Company. Tween is a registered trademark of ICI Americas, Inc. 14 5