HaloTag TM Technology: Cell Imaging and Protein Analysis

Transcription

1 Technology: Cell Imaging and Protein Analysis By G. Los, Ph. 1, R. Learish, Ph. 1, atasha Karassina, M.S. 1, Chad Zimprich, B.S. 1, Mark G. Mcdougall, Ph. 2, Lance P. Encell, Ph. 1, Rachel Freidman-hana, Ph. 1, Monika Wood, M.S. 1, Gediminas Vidugiris, Ph. 1, Kris Zimmerman, B.S. 1, Paul tto, M.S. 1, Soshana Berhstock, Ph. 1, Dieter. Klaubert, Ph. 2, and Keith V. Wood, Ph. 1, 1 Promega Corporation, 2 Promega Biosciences, Inc. Interchangeable Labeling Technology in vitro A B (1) (2) de novo Interchangeable Labeling Technology Interchangeable Labeling Technology DA 1 3 pt2 Vector pfc8a pfc8k CMV Flexi Vector 5-60 alolink TM 1 TMR Ligand p65- C 1. p65- TMR Ligand elap65- p65-5µm TMR Ligand (3)3.7% 1µg/ml mouse Anti-βIII Tubulin Antibody G7121Alexa Fluor TM -488-conjugated goat-antimouse IgG Molecular Probes FV500TMR Alexa Fluor TM A. TMR B. Alexa Fluor TM -488 Alexa Fluor TM -488TMR Alexa Fluor TM -488TMR Interchangeable Labeling Technology Ligand 233kDa C pt2 Vector CMV5'- in vitrot7 SV40 late (3)pFC8A pfc8k CMV Flexi Vector Factor Xa 4, 7 D 4883TA 12 Prometech Journal umber

2 alotag TMR Ligand -terminus Functional Reporter + alotag TMR Ligand 555 Ex /585 Em C CI 3 6 MW = C Reactive Linker C-terminus alotag diacfam Ligand 494 Ex /526 Em (after hydrolysis) C CI 10 MW = TMR Ligand Connolly surface TMR Ligand 4787TB alotag Coumarin Ligand 353 Ex /434 Em C CI 2 5 MW = 439 alotag Biotin Ligand C CI 3 4 S MW = S 2 1) 2) TMR diacfam Coumarin 3 GST- TMR Ligand in vitro TMR- (5) - (6) Technical Manual #TM260 Ligand 1 Monster Green Fluorescent Protein (hmgfp) 21 hmgfp C3 [(LS) 3] Cα-tubulin ela - (LS) 3 hmgfp-α-tubulin -α-tubulin hmgfp-(ls) 3 -TMR Ligand Coumarin Ligand 4 -(LS) 3 coumarin TMR 4AC alotag PEG-Biotin Ligand C CI 4 9 S MW = 697 4A TMRCoumarin hmgfp-α-tubulin 4BD -α-tubulin TMR 4E α-tubulin hmgfp-(ls) 3 hmgfp GFP elac-k1 Prometech Journal umber S Spacer 3. PEG-Biotin Ligand 5515MA 13

3 A. B. 0 minutes 4 hours 4 hours 40 minutes E. F. 5 hours 4990TA 6 hours 4. hmgfp -(LS) 3 hmgfp-α-tubulina-d -αtubulinhmgfp-(ls) 3E FeLa 24 -(LS) 3 25µM Coumarin LigandA B5µM TMR Ligand C D -α-tubulin 5µM TMR LigandE F 1537 /5% C2 30AB Coumarin Ligand #31000 DAPI hmgfp #41001 FITC Chroma Technology Corp rca CCD environmental controls IX81 C-F2 TMR FITC 6 hours 40 minutes 7 hours 8 hours p65 κb F-κB LS nuclear localization sequencef-κb IκB F-κB (7-9) p p65- TMR Ligand TA 5. p65- ela p65- ela 5µM TMR Ligand (3) FV500TMR p65- -FLAG TMR Ligand diacfam Ligand Prometech Journal umber

4 A. B. A. B. E. F. 4986TA 6. p65- -FLAG- p65- -FLAGeLa 24 TMR Ligand 5µM, 37, 15 ; A 18 diacfam Ligand 1µM, 37, 30 ; B FV500 TMR FAM C, ; D, TMR FAM 5729TA 7. ela -α-tubulin diacfam A-CTMR D-F 1 0.1% Triton X-100 Anti-βIII-Tubulin mab 5,000, G Alexa Fluor TM 488 2A-CCy 3 2 D-F AD A diacfam D TMR BE βiii-tubulincf βiii-tubulin TMR diacfam Ligand 18 6B TMRFAM TMR Ligand diacfam Ligand FAM - Ligand ICC, 10 -α-tubulin TMR Ligand diacfam Ligand βiii-tubulin 1 Alexa Fluor TM 488Cy 3 2ICCeLa βiii-tubulin7 - α-tubulin ICC2 ligand treated control ligand treated control C UB B C UB B C UB B C UB B p65-ab IκB-Ab 8. (SA-PMP) p65- IκB p65- C-K125µM Biotin Ligand Z5481 SDS-PAGE anti-p65 antibody BD Bioscience, anti-iκb antibody BD Bioscience, C= cell lysate UB=unbound protein B=bound protein 4886TA Prometech Journal umber

5 - SDS-PAGE SDS-PAGE (10) C- K1p65- Biotin Ligand Streptavidin MagneSphere Paramagnetic Particle SDS-PAGE 8p65-8 p65- IκB 8 Interchangeable Labeling Technologyin vitro - SDS-PAGE 1. Giuliamo, K.A. and Taylor, L. (1998) Trends. Biotechnol. 16, Chapman-Smith, A. and Cronan, J.E. (1999) Trends Biochem. Sci. 24, alotag Interchangeable Protein Labeling Technology Technical Manual #TM240, Promega Corporation. 4. Urh, M. et al. (2005) Promega otes 92, Los, G. (2005) Cell otes 11, Quershi, M.. et al. (2001) J. Biol. Chem. 276, Ghosh, S., May, M.J. and Kopp, E.B. (1998) Ann. Rev. Immunol. 16, Karin, M., Yamamoto, Y. and Wang, Q.M. (2004) at. Rev. Drug Discov. 3, Burnstein, E and Duckett, S. (2003) Curr. pin. Cell Biol. 15, Los, G. et al. (2005) Promega otes 89, 2 5. alotag Interchangeable Labeling Technology Technical Manual, #TM260, Promega Corporation. alotag Interchangeable Labeling Technology halotag01 pt2 Vector 20 µg G ,000 pfc8a ( ) CMV Flexi Vector 20 µg C ,000 pfc8k ( ) CMV Flexi Vector 20 µg C ,000 TMR Ligand 30 µl G ,000 diacfam Ligand 30 µl G ,000 Coumarin Ligand 30 µl G ,000 Biotin Ligand 30 µl G ,000 PEG Biotin Ligand 30 µl G ,000 alolink TM Resin 2 ml G ,000 5 ml G , Prometech Journal umber