Abstract
Specific and targeted photoactivation of protein function inside cells, tissues, or whole organisms can be achieved with reversible inhibition of proteins by conjugation with photolabile protection compounds (“caging”). In vitro caging of proteins is thought to cause sterical or functional hindrance of amino acid side chains that are important for protein activity. Following the modification, the caged protein is introduced into the biological system and high-resolution irradiation ensures specific release of protein function in the desired areas. Here, I describe the entire caging procedure and highlight a few of the caveats of photoactivation in living cells.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kaplan JH, Hollis RJ (1980) External Na dependence of ouabain-sensitive ATP:ADP exchange initiated by photolysis of intracellular caged-ATP in human red cell ghosts. Nature 288(5791):587–589
Brieke C, Rohrbach F, Gottschalk A, Mayer G, Heckel A (2012) Light-controlled tools. Angew Chem Int Ed Engl 51(34):8446–8476. doi:10.1002/anie.201202134
Mayer G, Heckel A (2006) Biologically active molecules with a “light switch”. Angew Chem Int Ed Engl 45(30):4900–4921. doi:10.1002/anie.200600387
Marriott G (1994) Caged protein conjugates and light-directed generation of protein activity: preparation, photoactivation, and spectroscopic characterization of caged G-actin conjugates. Biochemistry 33(31):9092–9097
Cambridge SB, Davis RL, Minden JS (1997) Drosophila mitotic domain boundaries as cell fate boundaries. Science 277(5327): 825–828
Kossel AH, Cambridge SB, Wagner U, Bonhoeffer T (2001) A caged Ab reveals an immediate/instructive effect of BDNF during hippocampal synaptic potentiation. Proc Natl Acad Sci U S A 98(25):14702–14707. doi:10.1073/pnas.251326998
Humphrey D, Rajfur Z, Imperiali B, Marriott G, Roy P, Jacobson K (2007) Introduction of caged peptide/protein into cells using microinjection. CSH protocols 2007:pdb prot4659. doi:10.1101/pdb.prot4659
Humphrey D, Rajfur Z, Imperiali B, Marriott G, Roy P, Jacobson K (2007) Introduction of caged peptide/protein into cells using bead loading. CSH protocols 2007:pdb prot4658. doi:10.1101/pdb.prot4658
Butcher EC, Scollay RG, Weissman IL (1980) Direct fluorescent labeling of cells with fluorescein or rhodamine isothiocyanate. II. Potential application to studies of lymphocyte migration and maturation. J Immunol Methods 37(2): 109–121
Cambridge SB, Geissler D, Calegari F, Anastassiadis K, Hasan MT, Stewart AF, Huttner WB, Hagen V, Bonhoeffer T (2009) Doxycycline-dependent photoactivated gene expression in eukaryotic systems. Nat Methods 6(7):527–531
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Cambridge, S. (2014). Modification of Purified Proteins with Photochemical Protection Compounds for High-Resolution Photoactivation of Protein Function In Vitro and In Vivo. In: Cambridge, S. (eds) Photoswitching Proteins. Methods in Molecular Biology, vol 1148. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0470-9_2
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0470-9_2
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0469-3
Online ISBN: 978-1-4939-0470-9
eBook Packages: Springer Protocols