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Instructions rAVCMV–LifeAct®–TagRFP
LifeAct® is a 17 amino acids long fragment of a protein originating from Saccharomyces cerevisiae,which comprises an actin–binding domain. This marker can be used in various eukaryotic cellsto stain filamentous actin (F–actin). Used in living cells it is perfectly labeling the highly dynamicF–actin and moreover, does not interfere with cellular processes.
Please read the following Application Note for more detailed handling information:
Application Note 28 ”Protocol for Adenoviral Transduction of Human Cells“: This AN provides an example protocol for aboosted transduction of HUVEC in µ-Slide 8 Well.
Vector Description
Adenoviral vectors have become major tools for transientgene transfer and protein expression in cells that havea low transfection efficiency with standard transfectionmethods. Various dividing and non–dividing mammaliancell types can be infected with adenoviral vectors for highexpression of a recombinant protein. After transduction,the virus stays epichromosomal, meaning not integratinginto the host genome.
rAVCMV–LifeAct®–TagRFP is a E1/E3–deleted, human
Adenovirus (Serotyp 5) comprising LifeAct®–TagRFP fu-sion protein. The vector can be used for fluorescent label-ing of the actin cytoskeleton in various living cells withspecial focus on primary cells. TagRFP codon usage isoptimized for high expression in mammalian cells, i.e.humanized. Actin–binding domain of the yeast proteinAbp140 is fused to the TagRFP N–terminus.
For strong expression of the fusion protein in mammaliancells the vector contains the cytomegalovirus (CMV) pro-moter. SV40 polyadenylation signals (SV40 poly A) directproper processing of the 3’ end of the reporter mRNA.
Vector Map
ITR: Inverted terminal repeatPCMV : Cytomegalovirus promoterTransgene: LifeAct®–TagRFPpolyA: SV40 polyadenylation signal
Ad5 Genome: Adenovirus serotype 5 genome(E1/E3–deleted)
Specifications
TagRFP Fluorescence
Exmax 555 nmEmmax 584 nmFind more information on www.evrogen.com.
Packaging and Storage
Amount 100 µlConcentration 1 × 10 10 IU/mlShipping conditions dry iceStorage conditions -80°C*Shelf life Under proper storage condi-
tions as indicated on vial.*Avoid repeated freeze and thaw cycles as this leads to strong decreasein viral titer.
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Instructions rAVCMV–LifeAct®–TagRFP
Transduction of Mammalian Cells in vitro
The transduction efficiency of adenoviral particles variesin dieffernt cell types and therefore, has to be determinedin advance for obtaining the optimal transduction rate. Arange of 10-200 MOIs (multiplicity of infection) is used formost cell types. However, up to 1000 MOI may be neces-sary for lymphoid cell types.
Note:
ibiBoostTM Adeno (50301) enhances adenoviral trans-duction of most cell types. If working with a cell typefor the first time, only one well should be used asibiBoostTM Adeno control to determine cell sensitiv-ity.
Safety Consideration:
Note, that you will be working with samples contain-ing infectious virus. Follow the recommended NIHguidelines for all materials containing BSL–2 organ-isms. Always wear gloves, use filtered tips and workunder a biosafety hood.
Selected References
D.l R. Overby, E. H. Zhou, R. Vargas-Pinto, R. M. Pedrigi, R. Fuchshofer, S. T. Braakman, R. Gupta, K. M. Perkumas, J. M.Sherwood, A. Vahabikashi, Q. Dang, J. H. Kim, C. R. Ethier, W. D. Stamer, J. J. Fredberg, and M. Johnson. Alteredmechanobiology of schlemm’s canal endothelial cells in glaucoma. Proceedings of the National Academy of Sciences, 2014.doi: 10.1073/pnas.1410602111.
J. Riedl, A. H. Crevenna, K. Kessenbrock, J. H. Yu, D. Neukirchen, M. Bista, F. Bradke, D. Jenne, T. A. Holak, Z. Werb,M. Sixt, and R. Wedlich-Soldner. Lifeact: a versatile marker to visualize f-actin. Nature Methods, 5(7):605–607, 2008. doi:10.1038/nmeth.1220.
K. Sliogeryte, S. D. Thorpei, Z. Wang, C. L. Thompson, N. Gavara, and M. M. Knight. Differential effects of LifeAct-GFPand actin-GFP on cell mechanics assessed using micropipette aspiration. Journal of Biomechanics, 2016. doi: 10.1016/j.jbiomech.2015.12.034.
R. Vargas-Pinto, H. Gong, A. Vahabikashi, and M. Johnson. The effect of the endothelial cell cortex on atomic force mi-croscopy measurements. Biophysical Journal, 105, 2013. doi: 10.1016/j.bpj.2013.05.034.
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Instructions rAVCMV–LifeAct®–TagRFP
Ordering Information
LifeAct® Plasmids
Cat. No. Description Amount
60101 pCMV–LifeAct®–TagGFP2: plasmid, lyophilized 20 µg60102 pCMV–LifeAct®–TagRFP: plasmid, lyophilized 20 µg60106 pCAG–LifeAct®–TagGFP2: plasmid, lyophilized 20 µg60107 pCAG–LifeAct®–TagRFP: plasmid, lyophilized 20 µg
LifeAct® Adenoviral Vectors
Cat. No. Description Amount
60121 rAVCMV–LifeAct®–TagGFP2: adenoviral vector, ready to use,1 × 1010 IU/ml
1 × 109 IU
60122 rAVCMV–LifeAct®–TagRFP: adenoviral vector, ready to use,1 × 1010 IU/ml
1 × 109 IU
LifeAct® Lentiviral Vectors
Cat. No. Description Amount
60141 rLVUbi–LifeAct®–TagGFP2: lentiviral vector, ready to use, 1 × 107 TU/ml 1 × 106 TU60142 rLVUbi–LifeAct®–TagRFP: lentiviral vector, ready to use, 1 × 107 TU/ml 1 × 106 TU
LifeAct® Cell Lines
Cat. No. Description Amount
40101 HT-1080 LifeAct®–TagGFP2: HT-1080 cells expressingLifeAct®–TagGFP2
5 × 105 cells
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Instructions rAVCMV–LifeAct®–TagRFP
Notice to Purchaser
Evrogen Fluorescent Proteins TagGFP2 and TagRFP
This product is for internal non–commercial research use only. No rights are conveyed to modify or clone the gene encoding fluorescent protein containedin this product. The right to use this product specifically excludes the right to validate or screen compounds. For information on commercial licensing,contact Evrogen Licensing Department, email: [email protected].
LifeAct® Product and its Variants
Use of these products and product applications are covered by european and foreign patents and patents pending.
All names containing the designation ® are registered with the Office for Harmonization in the Internal Market and the U.S. Patent and Trademark Office.
Not–For–Profit Entities: Orders may be placed in the normal manner by contacting your local representative or ibidi Customer Service at +49 (0)89/5204617 0. At its discretion, ibidi grants Not–For–Profit Entities a non–exclusive, personal, limited license to use this product for non–commercial life scienceresearch use only. Such license specifically excludes the right to sell or otherwise transfer this product, its components or derivatives thereof to thirdparties. No modifications to the protein coding sequence may be made without express written permission from ibidi. Any other use of this productrequires a license from ibidi. For–Profit Entities wishing to use this product are required to obtain a license from ibidi. For license information, pleasecontact a licensing representative by email at [email protected].
For research use only!
Further technical specifications can be found at www.ibidi.com. For questions and suggestions please contact us by e-mail [email protected] by telephone +49 (0)89/520 4617 0. All products are developed and produced in Germany.© ibidi GmbH, Am Klopferspitz 19, 82152 Martinsried, Germany.
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