Contents イントロダクション 2 NanoLuc アプリケーション 5 タンパク質の安定性 5 タンパク質間相互作用 6 タンパク質発現検出(HiBiT) 10 タンパク質 -リガンド相互作用 12 HaloTag アプリケーション 13 分子間相互作用 13 イメージング 16 タンパク質

Transcription

1 Contents イントロダクション 2 NanoLuc アプリケーション 5 タンパク質の安定性 5 タンパク質間相互作用 6 タンパク質発現検出(HiBiT) 1 タンパク質 -リガンド相互作用 12 アプリケーション 13 分子間相互作用 13 イメージング 16 タンパク質精製 21 タグ融合タンパク質の発現 (How To) 23

2 タンパク質の を 生 た細胞で の 分析 の 分 解析 ックス解析 の 解 の プ ーチと の ータの 解 タンパク質の の 解 の のップ の タンパク質 解析 化 質 分析 プランSPR の の タンパク質 解析 のタンパク質 と と 5 ページ Nano-Glo Luciferase Assay System 6 ページ NanoBRET Nano-Glo Detection System 12 ページ NanoBRET Target Engagement Assay 融合 NanoBRET 618 Ligand NanoLuc 融合タンパク質 蛍光 NanoBRET Tracer NanoBRET PPI System タンパク質 : タンパク質相互作用 蛍光 NanoBRET Target Engagement Systems タンパク質 : リガンド相互作用 融合タンパク質 NanoLuc Fusion Vectors VHL Ub Ub Ub Ub Degradation Ub + タンパク質の安定性 NanoLuc 融合タンパク質作製方法 ベクター構築 & トランスフェクション ゲノム編集 HIF 通常酸素状態 ( 低発光値 ) VHL NLuc HIF NLuc NanoLuc Luciferase 23 ページ HIF VHL NLuc NLuc HIF 低酸素状態 ( 高発光値 ) NanoLuc Luciferase Fusions NanoLuc Binary Technologies SmBiT (11aa) LgBiT (156aa) Fusion Vectors 融合タンパク質作製方法 ベクター構築 & トランス フェクション 23 ページ クロマチン免疫沈降 13 ページ HaloCHIP System 14 ページ タンパク質 :DNA 相互作用 プルダウンアッセイ タンパク質 : タンパク質相互作用 Mammalian Pull-Down System タンパク質精製 HaloLink Resin 21 ページ Protein Purification System 2

3 た ー NanoLuc クノ ー のタンパク質 の と タ クノ ー GFP His タ と のタ レベルの分 可 分 の タ HiBiT 11 ノ タンパク質 の と のタンパク質 解析 と の イ NanoLuc クノ ー と タンパク質 ライ ルイ ー ン CRISPR ゲノム の プ ーション 9 ページ タンパク質 : タンパク質相互作用 Nano-Glo Live Cell Assay System LgBiT 11 ページ Extracellular Nano-Glo HiBiT Detection System ( 細胞非溶解 ) NanoBiT PPI System NanoBiT Luciferase 培養液中への分泌 SmBiT Fusion Vectors 低親和性 ( 補助的な結合 ) 用途 : タンパク質間相互作用 HiBiT (11aa) 高親和性 ( 自発的な結合 ) 用途 : 発光タグ LgBiT Fusion Vectors HiBiT 融合タンパク質作製方法 ベクター構築 & トランスフェクション ゲノム編集 23 ページ HiBiT 融合細胞表面タンパク質 HiBiT 融合細胞内タンパク質 何らかの処理を行った後のタンパク質レベルの変化 HiBiT 融合分泌タンパク質 LgBiT 1 ページ Lytic Nano-Glo HiBiT Detection System ( 細胞溶解 ) 1 ページ Nano-Glo HiBiT Blotting System タンパク質の局在 動態 生細胞蛍光イメージング 蛍光タンパク質とのマルチプレックス解析 分解過程の可視化 パルスチェイス 蛍光リガンド 16 ページ FACS HiBiT 融合タンパク質のプロセシング 発現の変化 その他を LgBiT/HiBiT ブロッティングで確認 Ligands 蛍光 SDS-PAGE 融合タンパク質の発現量 プロセシングなどの変化を確認 3

4 NanoLuc [Oplophorus gracilirostris] 19kDa Rhodococcus rhodochrous DhaA 1 Coelenterazine Furimazine BRET 2 HiBiT NanoBiT NanoLuc 11 HiBiT SmBiT 18KDa LgBiT NanoLuc LgBiT HiBiT SmBiT NanoBiT 標的タンパク質 TEV Site HaloTag タンパク質 イメージング用リガンド Coumarin Ligand TMR Ligand Oregon Green Ligand Alexa Fluor 488 Ligand プルダウン 精製用リガンド Biotin Ligand HaloLink TM HaloLink TM Magnetic Resin Beads Protein Detection PPI Asp16 共有結合 機能性レポーター分子 未標識リガンド high affinity low affinity Asp16?? Amine Ligand Succinimidyl Ligand 11 a.a. HiBiT 17.6kDa LgBiT 11 a.a. SmBiT 19kDa LgBiT 17.6kDa SmBiT 11 Kd=19µl LgBiT 17.6kDa HiBiT 11 Kd=7 pm NanoLuc Luciferase NanoBiT PPI System HiBiT 19kDa 1 BRET BRET NanoLuc 17.6kDa LgBiT 33kDa In vivo 4

5 NanoLuc Stability Sensor NanoLuc HIF1A NRF2 NanoLuc Stability Sensor ready-to-use NanoLuc 2 HIF1A NRF2 HIF1A Vector System NRF2 Vector System HIF1A NRF2 HIF1A Vector System HIF1A NRF2 Vector System NRF2 HIF1A NRF2 NanoLuc C CMV Transfection Carrier DNA NRF2 Vector System NRF2 pkeap1 pnlf1-hif1a CMV/neo Vector 2 µg N1381 pnlf1-nrf2 CMV/neo Vector 2 µg N1391 NanoLuc VHL Ub Ub Ub Ub Degradation Ub + Nano-Glo Luciferase Assay System HIF 通常酸素状態 ( 低発光値 ) HIF1A VHL NLuc HIF NLuc HIF VHL NLuc NLuc HIF 低酸素状態 ( 高発光値 ) 1 ml N111 1 ml N ml N ml N115 SDS-PAGE Nano-Glo In-Gel Detection NanoLuc NanoLuc Nano-Glo In- Gel Detection Reagent PAGE 15 SDS-PAGE SDS NanoLuc 25% 2 2 NanoLuc Nano-Glo In-Gel Detection System A. kda Carrier DNA NEK7 STK32A CAMKK2 RPS6KA5 STK1 NEK7 + CAMKK2 + STK1 STK32A + RPS6KA5 All constructs B. kda M Carrier DNA NEK7 STK32A CAMKK2 RPS6KA5 STK1 M NEK7 + CAMKK2 + STK1 STK32A + RPS6KA5 All constructs SDS-PAGE NanoLuc Nano-Glo In-Gel Detection System HEK293 SDS-PAGE 5 NanoLuc NEK7, STK32A, CAMKK2, RPS6KA5, STK1 HEK293 CMV DNA 1 µl A. NanoLuc Nano-Glo In-Gel Detection System 3 B. Comassie Nano-Glo In-Gel Detection System 1 ml N MA 5

6 NanoLuc 正確な定量のために タンパク質 - タンパク質相互作用の定量 BRET 生物発光共鳴エネルギー移動 A. NanoBRET NanoLuc 生細胞 アッセイ HaloTag タンパク質 A タンパク質 B 生細胞リアルタイム PPI モニタリング 生物発光共鳴エネルギー移動 Bioluminescence resonance energy transfer は 生細胞におけるタンパク間の直接的な相互作用を検出す る手法として幅広く用いられています 類似の原理である FRET Fluorescence resonance energy transfer を用いた手法に比べ 励起光を 当てる必要がないので バックラウンドがきわめて低く 励起光による細胞へのダメージを最小限に抑えることができます SmBiT 11 amino acidsは従来のウミシイタケルシフェラーゼを用いた 発光源に高レベル発光酵素 NanoLuc を用いた NanoBRET system BRET に比べ より高 LgBiT SmBiT kda 17.6 い発光値と安定性 強固性を持ち また LgBiT 11 amino acids HaloTag NanoBRET 618 fluorescent Ligand で標識された HaloTag タンパク質をエネルギー転 17.6 kda 移アクセプターとして利用することで従来の BRET に比べ優れたシグナル / バックグラウンド比が得られます 生細胞でのリアルタイム解 構造的相補性による発光シグナル 析 及びハイスループットスクリーニングに最適です B. タンパク質 A タンパク質 B A. Protein A Protein B O HaloTag ベクターを導入した細胞内で発現した ligand 青色 FKBPタンパク 質 A NanoLuc 融合タンパク質と オレンジ色 に低分子蛍光 618リガンドが 結合したタンパク質 B HaloTag 融合タンパク質が近接すると BRET が NLuc FRB 起こる 低分子蛍光 618リガンドはタンパク質発現後に細胞へ添加する Rapamycin 細胞透過性の蛍光試薬 BRET Fluorescence 618 Ligand HL: HaloTag NanoBRET 618 Ligand B. + substrate NanoBRET システムの原理 A. O O O BRET による蛍光シグナル : HaloTag protein Donor signal NanoBRET emission NanoLuc luciferase BRET energy transfer Background 1..9 FKBP FRB NLuc Relative Intensity.7 Acceptor 618nmEx = Ratio Donor 46nmEm.5 Acceptor signal.3 FKBP FRB NLuc TB FRB NLuc 12 61nm 61nm BRET Frb-NLuc/FKBP- Frb- RLuc8/FKBP-YFP 11 ligand Rapamycin.4 B. A..8.6 FKBP BRET , Rapamycin (nm) B Wavelength (nm) 11 新しい発光酵素 NanoLuc ピーク波長 46 nm と蛍光リガンド 618 Ligand ピーク 波長 618 nm により シグナルの増強とスペクトルの分離が大幅に改善され 従来 の FRET/BRET では得られなかったシグナル /ノイズ比を実現 Frb-NLuc/FKBP- Frb- RLuc8/FKBP-YFP 従来の 9 BRET アッセイと NanoBRET のパフォーマンス比較 Signal-to-Background Ratio BRET に最適なスペクトルの分離とシグナル強度 Ligand が 結 合した FKBP-HaloTag と FRB-NanoLuc 存 在 下 でラパ マイ HaloTag 7 シンを加えると 2 つの融合タンパク質が相互作用を起こし BRET を生じる 左図 6 と FRB-Rluc8 による BRET システムとのデータ比較 FKBP-YFP Vasopressin AVPR2.1 barr2-nl.1 1 AVPR- 1 Rapamycin (nm) 1,barr2-NL 12323MA AVPR2 AVPR- B BRET ratio (mbu) BRET (%) AVP A AVP (nm) 6 NanoBRET による細胞イメージング アルギニンバソプレシン受容体 2 AVPR2 とβ -アレスチンとの相互作用の BRET 解析 撮影は Olympus LV2を使用 Zeit (Sekunden) 12323MA NanoLuc O HL Signal-to-Background Ratio O NanoLuc

7 NanoLuc NanoBRET NanoBRET NanoLuc BRET Ratio 8 NanoBRET NanoBRET NanoBRET NanoBRET 8 1 Protein A Protein B NLuc NLuc NL A B NL A B A NL B A NL B A A A A B NL B NL NL B NL B BRET Ratio (mbu) A- + NL-B 5. -A + NL-B 14.3 A- + B-NL 9.1 -A + B-NL 2.3 -B + A-NL 3. -B + NL-A 5.7 B- + A-NL 7.2 B- + NL-A N C 8 NanoBRET BRET Ratio MCS Flexi Flexi NanoBRET PPI MCS Starter System 1 N1811 NanoBRET PPI Flexi Starter System 1 N1821 NanoLuc - Positive Control p53- NanoLuc -MDM2 PPI Control Pair NanoBRET Positive Control 2 2 µg N1581 NanoBRET PPI Control Pair p53, MDM2 2 2 µg N NanoBRET Interaction Assay BRD 4/Histone H3.3 1 N183 BRD 9/Histone H3.3 1 N184 BRPF1/Histone H3.3 1 N186 cmyc/max 1 N187 KRas/BRaf 1 N188 BRD 4/Histone H4 1 N189 BRD 9/Histone H4 1 N19 BRPF1/Histone H4 1 N191 NanoLuc NanoBRET Nano-Glo Detection System 2 N N N1663 GloMax Discover NanoBRET UV/ BRET FRET 2 GloMax Discover System 1 GM3 7

8 NanoLuc - NanoLuc 2 NanoBiT NanoBiT PPI NanoLuc Binary Technology NanoBiT NanoLuc Large BiT LgBiT Small BiT SmBiT LgBiT SmBiT タンパク質 A タンパク質 B LgBiT SmBiT 構造的相補性による発光シグナル NanoBiT A LgBiT B SmBiT NanoBiT(% signal decrease) ISO PRO FSK 1 NanoBiT Time(min) RLU RLU NanoBiT Firefly [Rapamycin](nM) [Rapamycin](nM) NanoBiT Firefly RLU RLU NanoBiT SmBiT-CA:LgBiT-R2A PKA NanoBiT HEK293 ISO PRO FSK [FK56](µM) NanoBiT [Rapamycin](nM) NanoBiT FKBP FRB HEK293 FK56 sec 1 sec 46 sec 82 sec % relative response time(sec) ADRBR-114 / β - HeLa PPI 1 Olympus LV2 MCS Flexi Flexi NanoBiT PPI MCS Starter System 1 N214 NanoBiT PPI Flexi Starter System 1 N215 FKBP, FRB NanoBiT PPI Control Pair FKBP, FRB NanoBiT Nano-Glo Live Cell Assay System 2 2 µg N216 1 N211 1, N212 1, N213 8

9 NanoLuc NanoBiT NanoBiT LgBiT SmBiT NanoBRET 8 7 NanoBiT NanoBiT NanoBiT 8 1 NanoBiT NanoBRET GloMax Navigator NanoBiT GloMax Navigator System 1 GM2 NanoBRET NanoBiT BRET 2 kda NanoLuc 3 kda 11 SmBiT 18 kda LgBiT 2 %CV RLU 2 1 BRET Olympus LV2 Olympus LV2 1 Nano-Glo Live Cell Assay, Endurazine, Vivazine NanoLuc NanoLuc NanoBiT Nano-Glo Live Cell Assay System 1 NanoLuc NanoBiT NanoBRET NanoLuc Nano-Glo Live Cell Substrate Dilution Buffer Nano-Glo Live Cell Reagent 2 Endurazine Vivazine Luminescence (RLU) Nano-Glo Live Cell Assay System Vivazine substrate Endurazine substrate Time (hours) 1 N211 Nano-Glo Live Cell Assay System 1, N212 1, N213.1 ml N258 Nano-Glo Vivazine Substrate 1 ml N ml N ml N257 Nano-Glo Endurazine Substrate 1 ml N ml N2572 Nano-Glo Extended Live Cell Substrate Trial Pack Endurazine Vivazine.1 ml.2 ml N259 Nano-Glo Live Cell Assay System Endurazine Vivazine 9

10 NanoLuc Nano-Glo HiBiT Lytic Detection HiBiT Nano-Glo HiBiT Lytic Detection System HiBiT - - LgBiT Frimazine Nano-Glo HiBiT Detection Reagent HiBiT LgBiT HiBiT NanoBiT HiBiT Lytic reagent LgBiT HiBiT Lytic Detection System 14284MA RNA -seq HiBiT HIF1A CRISPR/Cas9 HiBiT Nano-Glo HiBiT Blotting HiBiT 1 pg 5 HiBiT pg LgBiT HiBiT 5 HiBiT HiBiT CRISPR/Cas9 GAPDH HiBiT 1 19 moles Separate HiBiT-tagged proteins in cell lysate using SDS-PAGE. Transfer proteins from gel to nitrocellulose membrane. 6 1fg 1ng Gel Membrane Membrane Add Nano-Glo HiBiT Blotting System reagents to the membrane to detect HiBiT-tagged proteins. HiBiT Blotting System LgBiT 5 Image the membrane. SDS-PAGE LgBiT A. B. Log 1 Background-Subtracted Band Intensity acted y minute exposure 2.4-minute exposure 14-second exposure Log 1 HiBiT Control Protein (moles) 21 hours at 4 C 1 hour at room temperature hours 14368MB

11 NanoLuc Nano-Glo HiBiT Extracellular Detection HiBiT HiBiT HiBiT Nano-Glo HiBiT Extracellular Detection Reagent HiBiT Nano-Glo HiBiT Detection Reagent LgBiT HiBiT LgBiT NanoBiT Nano-Glo HiBiT Extracellular Detection System LgBiT ADRB2 HiBiT G P C R Gs ADRB2 アゴニスト Isoproterenol Salbutamol Salmeterol Formoterol + LgBiT インターナリゼーション EC5 LgBiT HiBiT NanoLuc 基質 LgBiT Isoproterenol 5.9 nm 16% Salbutamol 161 nm 45% Salmeterol 1.4 nm 63% Formoterol 2.92 nm 16% β 2- adrenergic receptor ADRB2 4 β 2-adrenergic receptor ADRB2 5-1 分 2-1 分 HiBiT システム HiBiT Extracellular 検出試薬を添加 インキュベート 発光値読み取り HiBiT ELISA GPCR HiBiT ELISA GPCR の N 末端にタグをつけ 細胞表面で発現 アゴニスト処理 細胞固定化 ブロッキング 抗タグ抗体処理 洗浄 標識 2 次抗体処理 洗浄 発色基質を添加してインキュベート 吸光度読み取り ELISA 法 2 時間 1 時間 1~3 分 5-6 時間 1 ml* N33 Nano-Glo HiBiT Lytic Detection System 1 ml N ml N35 Nano-Glo HiBiT Blotting System 1 ml** N241 1 ml* N242 Nano-Glo HiBiT Extracellular Detection System 1 ml N ml N2422 HiBiT Control protein 1 µl N31 *1ml 96 1 **1ml 1 11

12 NanoLuc HiBiT Intracellular Detection HiBiT HiBiT HiBiT LgBiT Nano-Glo Live Cell Assay Reagent 9 2 LgBiT HiBiT Nano- Glo Live Cell Assay Reagent HiBiT Exosome 融合 感染 本アプリケーションでは 2 つの細胞にそれぞれ LgBiT HiBiT を発現させる LgBiT HiBiT NanoLuc 基質 LgBiT/HiBiT CMV LgBiT Vector CMV -LgBiT Vector CMV -HiBiT Vector LgBiT HEK293 HEK293 LgBiT Stable Cell Line - BRET NanoBRET Target Engagement Assay NanoLuc NanoBRET Target Engagement TE Assay 4 NanoLuc NanoLuc NanoLuc NanoBRET System 6 NanoLuc BRET NanoLuc NanoLuc NanoLuc NanoBRET Target Engagement Intracellular HDAC Assay HDAC NanoBRET Target Engagement Intracellular BET BRD Assay BET BRD NanoBRET TE Intracellular Kinase Assay 12 2 K-4 K-5 BRET Nluc NanoBRET TE Assay NanoLuc NanoBRET Nluc NanoBRET Nluc NanoLuc luciferase Fluorescent tracer Test compound Target protein HDAC NanoBRET TE Intracellular HDAC Assay NanoLuc HDAC6 NanoBRET TE Intracellular HDAC Complete Kit NanoLuc HDAC1, HDAC2, HDAC3, HDAC6, HDAC1 HDAC6 CD2 1 N28 1, N281 1, N217 HDAC NanoBRET TE Intracellular HDAC Detection Reagents 1, N29 BET BRD NanoBRET TE Intracellular BET BRD Assay NanoLuc BRD4 NanoBRET TE Intracellular BET BRD Complete Kit NanoLuc HDAC1, BRD2, BRD3, BRDT, BRD4 BRD2 BD1, BRD2 BD2, BRD4 BD1, BRD4 BD2 1 N213 1, N2131 1, N218 BET BRD NanoBRET TE Intracellular BET BRD Detection Reagents 1, N214 1 N252 NanoBRET TE Intracellular Kinase Assay, K-4 1, N2521 1, N254 1 N25 NanoBRET TE Intracellular Kinase Assay, K-5 1, N251 1, N253 NanoLuc 12

13 -DNA HaloCHIP Assay ChIP HaloCHIP System /DNA Transfection ChIP fusion construct TF protein TF Iranscription tactor HL HaloLink Resin DNA DNA HaloLink Resin /DNA TF HL HaloCHIP Blocking Ligand DNA ChIP HaloLink Resin DNA HaloCHIP ChIP HL TF TF HL HL HaloCHIP Blocking Ligand TF HaloLink Resin days HaloCHIP DNA S/B ChIP 4 5 / A/G DNA S/B HL TF Sample DNA HL DNA 4 2,62 bp CREB ChIP-chip enrichment log2 ratio CREB enrichment CREB Halo-chip enrichment METTL4 CRE half site NDC8 CRE full site 9671MA log2 ratio CREB enrichment ALS2 1,125 bp CREB ChIP-chip enrichment CREB Halo-chip enrichment half site CRE full site 967MA HaloCHIP ChIPDNA HaloCHIP CREB:DNA Nimblegen promoter tiling array Hartzell, D. D.; Trinklein, N. D. et al. 29 A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays, BMC Genomics, 1: 497 HaloCHIP System HaloCHIP System ChIP DNA HaloCHIP System 2 G941 * HaloCHIP Blocking Ligand 3µl HaloLink Resin 2ml High Salt Wash Buffer 25ml Mammalian Lysis Buffer 4ml Nuclease-Free Water 15ml Reversal Buffer 8ml 13

14 - Pull-Down Assay bait GST HaloLink HaloLink bait/prey SDS-PAGE HaloTag vs GST bait/prey = 1% input fusion Protein HaloLink TM supernatants GST Resin 1min 24hr 1min 24hr GST GST GST-Fos (prey) HaloLink pull-down Jun- (bait) Control GST pull-down GST-Fos (bait) Control Jun- Fusion (prey) 共有結合 HaloLink TM GST GST Resin 弱い結合 25µl [ ] HaloLink Resin GST -GST 16µg PBS 4SDS-PAGE -GST 1% HaloLink 24 -GST GST Resin 1 -GST AEBSF HaloLink 5612TA Protease Inhibitor Cocktail AEBSF prey GST* * prey 6 Fos* Jun* bait Jun Fos bait His GST Flag GST Model Serine Cysteine Aspartate Metallo- Aminopepridases HaloLink TM GST Resin Promega * 35 S 標識タンパク質 Roche-cOmplate BD-BaculoGold HaloLink Resin GST GST-c-Fos c-jun- bait prey ³S - 2 HaloLink Resin GST- : HaloLink GST GST-c-Fos c-jun- : HaloLink GST PMSF Aprotinin EDTA Omplete is a trademark of Hoffmann-La Roche. BaculoGold is a trademark of Becton, Dickinson and Company. 1422MA 14

15 in vivo bait/prey = in vivo Mammalian Pull-Down and Labeling System TMRDirect Ligand Control Vector CMV T7 SP6 RNA p65 Ab No Ab p65- Cont POI POI POI HaloLink Resin p15 p1 POI Rel A POI Rel B C-Rel IκBα IκBb IκBe Mammalian Pull-Down Assay HeLa p65 - Mammalian Pull-Down Assay Mammalian Pull-Down Assay p65 22 Mammalian Pull-Down and Labeling System 24 G65 Mammalian Pull-Down System 24 G654 Complete Pull-Down System 1 G659 bait Mammalian Pull-Down Assay Control Vector 2 µg G6591 Protease Inhibitor Cocktail, 5X 1 ml G6521 Mammalian Lysis Buffer 4 ml G9381 HaloLink Resin 1 ml G1914 G65 G654 HaloLink Resin 5ml Protease Inhibitor Cocktail, 5X 1ml Mammalian Lysis Buffer 1ml 1X TBS Buffer 25ml SDS Elution Buffer 1.3ml 22 15

16 1 GFP - Non-Wash min 3min (24-48 hrs) Labeling hrs Labeling Cells Ligands Wash Media replacement Non-Wash A. C. E. B. D. F. 499TA hmgfp - NLS 3 hmgfp-a-tubulin A-D -a-tubulin hmgfp- NLS 3 E F HeLa 24 - NLS 3 25µM Coumarin Ligand A B 5µM TMR Ligand C D -a-tubulin 5µM TMR Ligand E F /5% CO2 3 A B Coumarin Ligand #31 DAPI hmgfp #411 FITC Chroma Technology Corp Orca CCD environmental controls IX81 C-F 2 TMR FITC Non-Wash TMRDirect R11Direct Ligand - "Non-Wash " ICC GFP A B "No-wash" / U2OS R11Direct Ligand A. B: DIC C p65- TMR Ligand HeLa p64- TMR Ligand 3.7% 1µg/ml mouse Anti- β III Tubulin Antibody G7121 Alexa Fluor -488-conjugated goat-antimouse IgG (Molecular Probes) FV5 A. TMR B. Alexa Fluor -488 C. Alexa Fluor -488 TMR D. Alexa Fluor -488 TMR D 4883TA 16

17 35 S Met 18 Biotin Ligand G8281 A Control TNF- Lactacystin + TNF- min 3min 6 min 12 min B Percent of initial fluorescence signal Control Lactacystin TNF- α TNF- α Time (min) TNF- α I κ B- A I κ B- HEK293 TMR 5 µm TNF- α 1 ng/ml) TNF- α 3 1 µm Lactacystin B FV5 1 Janelia Fluor FACS Janelia Fluor Janelia Fluor 1 No-Wash Janelia Fluor Grimm, J.B. et al. (215) A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat. Methods 12, Normalized Fluorescence 1..5 A B Ligand Janelia Fluor 549 Janelia Fluor Wavelength (nm) Excitation maximum (dotted lines) Emission maximum (solid lines) 549nm 571nm 646nm 664nm 553MD U2OS U2OS 2 nm Janelia Fluor 549 Ligand A Janelia Fluor 646 Ligand B 15 Janelia Fluor 549 Ligand 561 nm Janelia Fluor 646 Ligand637 nm Janelia Fluor Ligand Janelia Fluor is a registered trademark of Howard Hughes Medical Institute. 17

18 & / SDS-PAGE 1 A B A B A B 1 /, B: - A. B. cell-impermeant cell-permeant kda C. D TA A. β 1 Integrin- / Alexa Fluor 488 Ligand TMR Ligand B. β 1 Integrin- HEK293 TMR Ligand 2 Alexa Fluor 488 Ligand 3 Alexa Fluor 488 Ligand TMR Ligand 4 1 SDS-PAGE C. β 1 Integrin- HeLa Alexa Fluor 488 Ligand 1µM 3715 TMR Ligand 5µM 3715 Olympus FV5 D. 12 FACS FACS SDS-PAGE "Non-Wash " 16 FACS U2OS R11Direct Ligand Non-Wash 1%, 1% 1%R1 R11 R2 R3 2, FL1=R11, FL2=PI 7527TA 18

19 / Ex Em Janelia Fluor 549 Ligand NEW Janelia Fluor 646 Ligand NEW µg GA111 3 x 5 µg GA µg GA112 3 x 5 µg GA1121 Coumarin Ligand µl G µl G8581 Oregon Green Ligand µl G282 3 µl G281 diacfam Ligand µl G µl G8272 TMR Ligand µl G µl G8251 STELLA Fluor 65 Ligand nmol GCKA nmol GCKA38-2 STELLA Fluor 7 Ligand STELLA Fluor 72 Ligand ICG Ligand Alexa Fluor 488 Ligand µg G12 3 µl G11 Alexa Fluor 66 Ligand µl G µl G8471 No-wash R11Direct Ligand µl G3221 TMRDirect Ligand µl G2991 AcidiFluor Orange ph nmol GCKGC nmol GCKGC31-2 HPF ROS APF ROS CaSiR Ca Biotin Ligand PEG-Biotin Ligand 15 µl G µl G µl G µl G8591 O2 O4 Amine (O4) Ligand 5 mg P6741 Succinimidyl Ester (O4) Ligand 5 mg P6751 Iodoacetamide (O4) Ligand 5 mg P6771 Succinimidyl Ester (O2) Ligand 5 mg P1691 Amine (O2) Ligand 5 mg P6711 Technology Alexa Fluor 66, Janelia Fluor 646 Ligand TMR, Janelia Fluor 549, TMRDirect Ligand Alexa Fluor 488, Oregon Green, R11Direct Ligands Coumarin Ligand Cy 5 filter set (~64nmEx/7nmEm) TRITC filter set (555nmEx/58nmEm) FITC filter set (488nmEx/52nmEm) DAPI/AMCA filter set (345nmEx/445nmEm) 19

20 SDS-PAGE Ligand in vitro use SDS-PAGE SDS-PAGE SDS 955 RFU 8,, 6,, 4,, GST- r 2 =.98 2,, r 2 = pmol 62 kda 33 kda 26 pg GST 13 pg 66 pg 33pg 17 pg 85 pg 4 pg 2 pg 1 pg 5 pg GST- GST- FAM ligand TMR ligand Standard Protein 3 µg G4491 Ligand Anti- Monoclonal Antibody Anti- pab kda 22 M TA EZH2- fusion protein Anti- pab p65- HEK293 TMR Ligand Anti- pab Alexa Fluor 488-conjugated anti-rabbit-igg A. TMR Ligand B. Anti- pab C. Monoclonal Antibody EZH2-12kDa HEK293T Monoclonal Antibody 1 AP 2 Anti- pab 2 µg G9281 Anti- Monoclonal Antibody 2 µg G9211 2

21 Protein Purification Protein Purification System TEV HaloLink Resin TEV N HQ HisLink Resin pfn18a T7 Flexi Vector G2751 pfn18k T7 Flexi Vector G2681 Step 1 Step 2 Step 3 Step 4 POI POI POI HaloLink Resin HaloLink Resin - (POI: protein of interest) HaloLink Resin HQ- TEV Protease POI coding region HisLink Resin HisLink Resin TEV Protease TEV Protease POI POI POI Protein of interest Purification System TEV [ENLYFQ G] His HQ HQHQHQ TEV HisLink Frequency of soluble proteins (%) high solubility medium solubility GST MBP His cpka activity (units/µg) Corrected for relative purity Without correction GST MBP His 1 high solubility: >1µg/ml, medium solubility: <1µg/ml cpka PKA cpka ; 99%, GST; 77%, MBP; 5%, His; 98% GST MBP His GFP 33kDa 26kDa 4kDa.85kDa 27kDa IMAC Protein Purification System Sample Pack Protein Purification System 5 ml culture ml culture 25 G627 G628 Coomassie Coomassie Coomassie Coomassie Coomassie Single Step KRX Competent Cells, >1 cfu/µg ProTEV Plus G627 HaloLink Resin 1ml [25% slurry] HisLink Protein Purification Resin 1ml [5% slurry] TEV Protease for use with HisLink Resin 2µl 2 5 µl L32 1, u V611 8, u V612 21

22 Mammalian Protein Purification System HaloLink Resin Mammalian Protein Detection and Purification System G6795, G6799 TMRDirect Ligand HaloLink Resin >7mg/ml FLAG 1 >75% <.1% HaloTEV Protease HaloLink Resin PKCγ PI3Kγ POI POI FLAG 3xFLAG His 6 tag FLAG 3xFLAG His 6 tag kda HaloLink Resin HaloLink Resin (POI) protein HaloLink Resin HaloLink Resin HaloTEV Protease POI HaloTEV Protease POI Protein of Interest POI Mammalian Protein Purification System % Recovery % Purity TB 2 PKC γ PI3K γ FLAG His Ohana, R.F. et al. 211 HaloTag-based purification of functional human kinases from mammalian cells. Protein Expr. Purif. Apr; 76 2 : Mammalian Protein Purification System 1 G679 Mammalian Protein Detection and Purification System Mammalian Protein Detection and Purification System Sample Pack HaloTEV Protease 1 G G µl G661 8 µl G ml 5ml * G1912 HaloLink Resin 2.5ml 1ml * G1913 1ml 4ml * G ml 1ml * G1915 2µl 1ml * G7281 Magne Beads 1ml 5x1ml * G7282 * SDS SDS-PAGE TEV HaloLink Resin: : 45 ~165µm 7mg/ml HaloLink Magnetic Beads: : 1 ~125µm 1mg/ml 22

23 DNA ORF NanoLuc 1, ORF cdna > 4, OC The ORFeome Collaboration ORF 2 NanoLuc Flexi pfn31k pfc32k Flexi pfn21a 25 1, ORF ORF Flexi Vector DNA Flexi Clone 1 FHCxxxxx Flexi NanoLuc Clone 1 FNCxxxxxN C 1ng 1µg 5µg 1µg Flexi ORF In Vitro Flexi ORF Clone ORF NaoLuc HAC TrueSTABLE Cell HAC ORF HAC HAC 1 Flexi ORF Clone & HEK293 cell NanoLuc HiBiT N C PPI vs etc. CMV EF-1 SDS-PAGE 23

24 pn CMV-neo Vector Yes CMV N No Neo Amp G7721 ph6c His6 T7 Vector Yes T7, 6xHis C No Amp G831 Flexi Vector 2 µg NanoBRET 7 NanoBiT 8 Starter System NanoLuc HiBiT / pfn31a Nluc CMV-Hygro Flexi Vector Flexi CMV Nluc N No Hygro Amp N1311 pfn31k Nluc CMV-neo Flexi Vector Flexi CMV Nluc N No Neo Kan N1321 pfc32a Nluc CMV-Hygro Flexi Vector Flexi CMV Nluc C No Hygro Amp N1331 pfc32k Nluc CMV-neo Flexi Vector Flexi CMV Nluc C No Neo Kan N1341 pnlf1-n[cmv/hygro]vector Yes CMV Nluc N No Hygro N1351 pnlf1-c[cmv/hygro]vector Yes CMV Nluc C No Hygro N1361 pnlf1-secn[cmv/hygro]vector Yes CMV Nluc N IL-6 Hygro N1371 pfc37k HiBiT CMV-neo Flexi Vector Flexi CMV HiBiT No Neo Kan N2391 pfn38k HiBiT CMV-neo Flexi Vector Flexi CMV HiBiT No Neo Kan N241 pfn39k sechibit CMV-neo Flexi Vector Flexi CMV HiBiT IL-6 Neo Kan N2411 pbit3.1-n [CMV/HiBiT/Blast] Vector Yes CMV HiBiT No Neo Kan N2361 pbit3.1-c [CMV/HiBiT/Blast] Vector Yes CMV HiBiT No Neo Kan N2371 pbit3.1-secn [CMV/HiBiT/Blast] Vector Yes CMV HiBiT IL-6 Neo Kan N2381 pfc14a HaloTag CMV Flexi Vector Flexi CMV C No Amp G9651 pfc14k CMV Flexi Vector Flexi CMV C No Kan G9661 pfc15a CMVd1 Flexi Vector Flexi CMV d1 C No Amp G1611 pfc15k CMVd1 Flexi Vector Flexi CMV d1 C No Kan G161 pfc16a CMVd2 Flexi Vector Flexi CMV d2 C No Amp G1591 pfc16k CMVd2 Flexi Vector Flexi CMV d2 C No Kan G1571 pfc17a CMVd3 Flexi Vector Flexi CMV d3 C No Amp G1551 pfc17k CMVd3 Flexi Vector Flexi CMV d3 C No Kan G1321 pfn21a CMV Flexi Vector Flexi CMV N No Amp G2821 pfn21k CMV Flexi Vector Flexi CMV N No Kan G2831 pfn22a CMVd1 Flexi Vector Flexi CMV d1 N No Amp G2841 pfn22k CMVd1 Flexi Vector Flexi CMV d1 N No Kan G2851 pfn23a CMVd2 Flexi Vector Flexi CMV d2 N No Amp G2861 pfn23k CMVd2 Flexi Vector Flexi CMV d2 N No Kan G2871 pfn24a CMVd3 Flexi Vector Flexi CMV d3 N No Amp G2881 pfn24k CMVd3 Flexi Vector Flexi CMV d3 N No Kan G2981 pfc27a CMV-neo Flexi Vector Flexi CMV C No Neo Amp G8421 pfc27k CMV-neo Flexi Vector Flexi CMV C No Neo Kan G8431 pfn28a CMV-neo Flexi Vector Flexi CMV N No Neo Amp G8441 pfn28k CMV-neo Flexi Vector Flexi CMV N No Neo Kan G8451 pc CMV-neo Vector Yes CMV C No Neo Amp G7711 pfn18k HaloTag T7 Flexi Vector Flexi T7 N No Kan G2681 pfn18a T7 Flexi Vector Flexi T7 N No Amp G2751 pfn29k His6 T7 Flexi Vector Flexi T7, 6xHis N No Kan G8331 pfn29a His6 T7 Flexi Vector Flexi T7, 6xHis N No Amp G8261 pfc3k His6 T7 Flexi Vector Flexi T7, 6xHis C No Kan G8381 pfc3a His6 T7 Flexi Vector Flexi T7, 6xHis C No Amp G8321 ph6n His6 T7 Vector Yes T7, 6xHis N No Amp G7971 pfn19k HaloTag T7 SP6 Flexi Vector Flexi T7, SP6 N No Kan G1841 pfn19a T7 SP6 Flexi Vector Flexi T7, SP6 N No Amp G1891 pfc2k T7 SP6 Flexi Vector Flexi T7, SP6 C No Kan G1691 pfc2a T7 SP6 Flexi Vector Flexi T7, SP6 C No Amp G1681 TEV Flexi 25 24

25 CMV Deletion Series Sampke Pack G378 pfc14k, pfc15k, pfc16k, pfc17k, pfn21a, pfn21k, pfn22k, pfn23k, pfn24k 2 µg G65 G378 Flexi System, Entry/Transfer C864, Carboxy Flexi Enzyme Blend Sgf I and EcoICR I R191 ori Amp r cer pfn21a ( ) CMV Flexi Vector (564bp) SV4 late poly(a) signal Barnase PmeI CMV Enhancer/ Promoter intron SgfI TEV site T7 7 Open Reading Frame 7549MA Flexi Vectors - CMV Deletion Series Sampke Pack 9 2 µg G378 Cloning Starter System 1 G65 Firefly Luciferase Expression (RLU) 1,, 1,, 1, 1, 1, CHO HEK293 U2OS NIH3T3 HeLa CMV CMVd1 CMVd2 CMVd3 CMV CHO CMV CMV> CMV d1> CMV d2 > CMV d3 Flexi CMV Vectors Flexi T7(SP6) Vectors pfc14 CMV Intron T7 ORF 7 RBS pfc15, pfc16, pfc17 CMVd T7 SP6 ORF 7 pfn21 CMV Intron T7 7 ORF RBS pfn22, pfn23, pfn24 CMVd T7 SP6 7 ORF SV4pA SV4pA SV4pA SV4pA pfn18 pfn19 pfn2 T7 T7 RBS T7 7 ORF A3 T7 ter RBS SP6 7 ORF A3 T7 ter RBS SP6 ORF 7 A3 T7 ter 7678MA CMV Promoter NFκB SP1 CREB SP1 TATA T7 SP6 Cloned gene CMVd1 CMVd2 CMVd3 Flexi Flexi Sgf I Pme I EcoICRI Barnase Amp-Kan ORF N Terminal Amp Target Gene Barnase C Terminal Kan Amp Kan Flexi N C N Flexi C Flexi Barnase Target Gene, Barnase 4 Amp Kan Barnase Amp Amp Kan Kan 25

26 HiBiT HiBiT HiBiT HiBiT CRISPR / Cas9 HiBiT HiBiT 33 bases Web crrna RNA crrna + tracrrna Alt-R CRISPR-Cas9 System IDT HiBiT Donor DNA template 5' Exon Exon Exon 3' UTR 3' Single-Stranded Oligodeoxynucleotide 5' ~8nt 33nt HiBiT Sequence TGA ~8nt 3' Homology Arm HiBiT Stop Codon Homology Arm 14511MA Normalized Luminescence (RLU) ng CMV/HIF1A-HiBiT 5ng CMV/HIF1A-HiBiT.5ng CMV/HIF1A-HiBiT.5ng CMV/HIF1A-HiBiT 5ng PGK/HIF1A-HiBiT Endogenous HIF1A-HiBiT clone Log 1 [1,1-phenanthroline], M HeLa CMV PGK HIF1A-HiBiT CRISPR/Cas9 HiBiT 1, MA RNP DNA Nano-Glo HiBiT Lytic Detection System LgBiT HiBiT CRISPR / Cas9 HiBiT 4-1 min HiBiT HiBiT HiBiT Web HiBiT 1 oligo HiBiT Forward NNNNNXXXXXXNNNNNNXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXNNNNNNXXXXXXNNNNN Reverse NNNNNXXXXXXNNNNNNXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXNNNNNNXXXXXXNNNNN Linker HiBiT hibitquick_guide.pdf 26

27 ViaFect Transfection Reagent ViaFect Transfection Reagent /DNA 8, ViaFect Transfection Reagent Amaxa T1 protocol A. B. C. Luminescence (RLU) 6, 4, 2, log[tnfα], ng/ml 1215MA ViaFect ips Cellular Dynamics icell 96 A. icell ViaFect DNA 6:1 GFP 1 GFP B. icell ViaFect DNA 2:1 GFP 1 GFP C. icell ViaFect DNA 4:1 GFP 3 GFP Cellular Dynamics International 12645TA ViaFect TF-1 pgl4.32 luc2p/nf- κ B-RE/Hygro Vector NF- κ B ViaFect Transfection Reagent Amaxa Nuclefector II TNF α 6 Bio-Glo Luciferase Reagent.75ml E4981 ViaFect Transfection Rreagent 2.75ml E ml 24 5 HUVEC Yokota, Y. et al. (215) Endothelial Ca 2+ oscillations reflect VEGFR signaling-regulated angiogenic capacity in vivo. elife 4, e8817. Nakmura, T. et al. (214) Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation. J. Cell Sci. 127, Bethge, T. et al. (215) Sp1 sites in the noncoding control region of BK polyomavirus are key regulators of bidirectional viral early and late gene expression. J. Virol. 89, Takahasi, M. et al. (215) Normalization of overexpressed α -synuclein causing Parkinson s disease by a moderate gene silencing with RNA interference. Mol. Ther. Nucleic Acids 12, e241. Straka, E. et al. (216) Mercury toxicokinetics of the healthy human term placenta involve amino acid transporters and ABC transporters. Toxicology 34, Egusa, S.F. et al. (216) Classic cadherin expressions balance postnatal neuronal positioning and dendrite dynamics to elaborate the specific cytoarchitecture of the mouse cortical area. Neurosci. Res. 15, Peng, Y. et al. (216) AGE-RAGE signal generates a specific NF- κ B RelA barcode that directs collagen I expression. Sci. Rep. 6, Naujoks, J. et al. (216) IFNs modify the proteome of Legionella-containing vacuoles and restrict infection via IRG1-derived itaconic acid. PLoS Pathogens 12, e1548. Huang, Y. et al. (215) Specific tandem 3 UTR patterns and gene expression profiles in mouse Thy1 + germline stem cells. PLoS One 1, e Horita, H. et al. (216) Nuclear PTEN functions as an essential regulator of SRF-dependent transcription to control smooth muscle differentiation. Nat. Comm. 7,

28 NanoLuc Stability Sensors for Cell Signaling - NanoBRET 6 - NanoBiT 8 BRET RNA HiBiT HiBiT - - Nano-Glo Luciferase Assay Substrate Nano-Glo HiBiT Blotting System Nano-Glo HiBiT Lytic Detection System Nano-Glo HiBiT Extracellular Detection System NanoBRET Target Engatement Assay NanoBRET Target Engatement Assay NanoBRET TE Intracellular Kinase Assay - DNA HaloCHIP System 13 - Pull-Down System, NanoBRET 1 TMR Ligand 19 RNA DAP-Seq Mammalian Protein Purification System Mammalian Protein Purification System Mammalian Protein Purification System Magne Beads 22 cdna Flexi Clone 23 Hall, M.P., et al. (212) Engineered luciferase reporter from a Deep Sea Shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem. Biol. 7, Narvaez, A.J., et al. (217) Modulating protein-protein interaction of the mitotic Polo-like kinases to target mutant KRAS. Cell Chem. Biol. 24, Machleidt, T., et al. (215) NanoBRET A novel BRET platform for the analysis of protein-protein interaction. ACS Chem. Biol. 1, Miyakawa, K., et al. (217) The tumour suppressor APC promotes HIV-1 assembly via interaction with Gag precursor protein. Nature Comm. 8, Dixon, A.S., et al. (216) NanoLuc complementation reporter optimized for accurate measurement of protein interactions in cells. ACS Chem. Biol. 11, 4-8. Yoshida, T., Kakizuka, A. and Imamura, H. (216) BTeam, a novel BRET-based biosensor for the accurate quantification of ATP concentration within living cells. Sci. Reports 6, Yang, J. et al. (216) Coupling optogenetic stimulation with NanoLuc-based luminescence (BRET) Ca++ sensing. Nature Comm. 7, Sasaki M., et al (218) Development of a rapid and quantitative method for the analysis of viral entry and release using a NanoLuc luciferase complementation assay. Virus Res. 243, Tamura, T., et al (218) Characterization of Recombinant Flaviviridae Viruses Possessing a Small Reporter Tag. J Virol. 92, e Oh-Hashi, K., et al (217) Application of a novel HiBiT peptide tag for monitoring ATF4 protein expression in Neuro2a cells. Biochem Biophys Rep. 12, Robers, M.B., et al. (215) Target engagement and drug residence time can be observed in living cells with BRET. Nature Comm. 6, 191. Stoddart, L.A., et al. (215) Application of BRET to monitor ligand binding to GPCRs. Nature Meth. 12, Robers, M. B., et al. (215) A luminescent assay for real-time measurements of receptor endocytosis in living cells. Anal. Biochem. 489, 1-8. Vasta, J.D., et al. (218) Quantitative, wide-spectrum kinase profiling in live cells for assessing the effect of cellular ATP on target engagement. Cell Chem. Biol. 25, Hartzell, D. D., et al. 29 A functional analysis of the CREB signaling pathway using HaloCHIP-chip and high throughput reporter assays, BMC Genomics, 1, 497 Moriya, C., et al. (218) PRDM14 directly interacts with heat shock proteins HSP9 α and glucose-regulated protein 78. Cancer Sci. 19, Miyanaga, Y., et al. 29 Single-Molecule Imaging Techniques to Visualize Chemotactic Signaling Events on the Membrane of Living Dictyostelium Cells. Methods in Molecular Biology 571, Toyosima, C., et al. (213) Crystal structures of the calcium pump and sarcolipin in the Mg2+-bound E1 state. Nature 495, Yokoshi, M., et al. (214) Direct binding of Ataxin-2 to distinct elements in 3 UTRs promotes mrna stability and protein expression. Molecular Cell 55, Ohana, R. F., et al. (211) HaloTag-based purification of functional human kinases from mammalian cells. Protein Expr Purif. 76, Bartlett, A., et al. 217 Mapping genome-wide transcription-factor binding sites using DAP-seq. Nat Protoc. 12, Nagase, T., et al. 28 Exploration of human ORFeome: high-throughput preparation of ORF clones and efficient characterization of their protein products. DNA Res. 15, Tel / Fax prometec@jp.promega.com プロメガ株式会社本社 東京都中央区日本橋大伝馬町 マツモトビル Tel /Fax 大阪事務所 大阪市淀川区西中島 花原第 8 ビル 74 号室 Tel /Fax Web site 製品の仕様 価格については 218 年 11 月現在のものであり予告なしに変更することがあります 販売店 PK1811-1B