Cell Imaging. Localization: Robust protein labeling of live or fixed cells

Transcription

1 F eat u r es & B e n efits ptions Cell Imaging with the alotag platform Localization: Robust protein labeling of live or fixed cells Trafficking & Turnover: Directly observe proteins with one or two colors in live cells Cell sorting: Simple non-antibody based cell labeling with multiple fluorophores

2 S i m p l e P roto cols ptions Simple cell-labeling protocols Day 1: Plate cells Day 2: Introduce alotag genetic construct into cells using standard transfection techniques Day 3: Label cells with alotag of choice Wash unbound ligand from sample or Add no wash ligand of choice Fixed-cell imaging Live-cell imaging alotag Protein alotag Excitation light Image fixed cells or perform ICC. Fluorescence Image live cells or chase with spectrally distinct ligand prior to imaging. Proceed with other analyses (e.g., SDS-PAGE, fluoroimaging, Western blotting). 7531MA

3 M u lt iple l a b e l i n g ptions Multiple labeling options Intracellular labeling (cell-permeable) o-wash labeling alotag TMRDirect alotag R110Direct alotag R110Direct C + 2 C 2 Ex: 555nm Em: 585nm Ex: 502nm Em: 527nm Rapid labeling alotag TMR + 2 C Ex: 555nm Em: 585nm alotag diacfam alotag regon Green F F Ex: 494nm Em: 526nm Ex: 496nm Em: 516nm alotag Coumarin 2 Ex: 353nm Em: 434nm alotag Alexa Fluor S S C Ex: 494nm Em: 517nm Cell surface labeling (membrane-impermeable) 9719MA

4 ptions fluorescent labeling of alotag fusion proteins on-cytotoxic, stable labeling with many fluorophore options Cova l e n t, F lu o r escent L a b e l i n g 9665TA 9665TB 9665TC Expressed alotag fusion proteins can be localized to different cellular compartments ucleus alotag -LS 3 Mitochondria Mito-aloTag Cytosol p65-alotag Membrane alotag-ecs 4975TA 9771TA

5 Long-term imaging with stable alotag ligands ptions Lo n g-t e r m I m ag i n g 0 hours 24 hours R110Direct TMRDirect U2S cells stably expressing alotag-ls3 in presence of TMRDirect and R110Direct ligands. was added only once prior to imaging. o additional ligand was added at later time points. Even after 120 hours, the ligand was still bound and bright. 120 hours 9767MA

6 bserve protein trafficking ptions P rot e i n T r a f f i c k i n g i n r ea l-t i m e 0 min 10 min 25 min 45 min 110 min ela cells expressing p65-alotag labeled with TMRDirect. Watch the translocation of p65 into the nucleus and then back out. 4976TA Watch the video (imaged every 5 min for 120 min after treatment with TFa)

7 ptions Study protein trafficking using dual labeling Label with membraneimpermeable green Monitor protein trafficking over time Label with cell-permeable red Wash A. B. Image (A) Incubate 12h Image (B) D ua l L a b e l i n g Within 12 hrs the alotag fusion protein on the surface (green) had been internalized while the internal protein (red) protein translocated to the cell surface. 6347TE Svendsen, S. et al. (2008) Spatial separation and bidirectional trafficking of proteins using a multi-functional reporter. BMC Cell Biol, 9: 17.

8 Analyze protein turnover with Pulse-Chase analysis ptions Pulse: R110Direct Replace media 0 hours Chase: TMRDirect Replace media 24 hours p u l s e-c h a s e a n a lys i s 9757MA eural progenitor cells expressing alotag were first labeled with R110Direct ligand (green). Afterward, the cells were cultured in the presence of TMRDirect ligand (red) to label newly-synthesized proteins. Protein turnover was determined 24-hours later by visualizing the difference between green and red signal. In collaboration with Erin McMillan and ive Svendsen

9 Cell sorting (FACS) of alotag expressing cells ptions r2 r1 0% Labeled r2 r1 100% Labeled r1= R110Direct labeled cells r2 = PI positive (dead) cells r3 = Unlabeled cells FL r FL % Labeled FL r FL % Labeled Cells were sorted in presence of propidium iodide (viability assay). R2 area demonstrates that increased ligand concentration does not affect cell viability r2 r r2 r1 FL FL r r TA C e l l S o rting FL1 FL1 alotag ligands are non-toxic to cells

10 ptions M o r e I n format i o n References Los, G.V. et al. (2008) alotag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol., 3(6): uybrechts, S.J. et al. (2009) Peroxisome dynamics in cultured mammalian cells. Traffic 10(11): Svendsen, S. et al. (2008) Spatial separation and bidirectional trafficking of proteins using a multi-functional reporter. BMC Cell Biol. 9: 17. Yamaguchi, K. et al. (2009) Pulse-chase experiment for the analysis of protein stability in cultured mammalian cells by covalent fluorescent labeling of fusion proteins. Methods Mol. Biol. 577: rdering Information P R D U C T S i z e C ata l o g # alotag s various G8251, G8252 alotag Reactive s 5mg P6751, P1691 A nt i- a l ota g pa b 200µg G9281 alotag is a registered trademark of Promega Corporation. alolink, TMRDirect, and R110Direct are trademarks of Promega Corporation. Promega Corporation 2800 Woods ollow Road Madison, WI USA Telephone Promega Corporation All Rights Reserved Prices and specifications subject to change without prior notice Printed in USA 2/ Part #IS037