269
13. The residual amount of culture medium after aspiration gains
increasing significance with decreasing surface area. Therefore,
washing the wells with PBS in case of small well format is
recommended (i.e., 48- /96-well format).
14. Light intensities of 5 μmol/(m
2
s) or lower are strongly recommended, since they sufficiently trigger the optogenetic System,
while showing negligible phototoxicity.
15. Illuminate the cell culture lab by safelight LEDs (i.e., wavelength longer than 500 nm). Measure the wavelength of LEDs
to ensure that they will not excite the blue light-responsive
CRY2/CIBN system.
16. After the centrifugation the almost clear pellet may be hard to
detect. Make sure not to transfer it, since it will lower the quality of the Western blot.
17. When boiling samples use Eppendorf Safe-Lock tubes. This
will prevent accidental opening of the tubes during boiling,
which leads to uneven protein amounts between samples.
18. Do not process too many samples at once. In case of the
CRY2/CIBN system, the light-induced interaction of the
fusion proteins is limited when working under safe light.
Acknowledgments
This work was supported by the Excellence Initiative of the German
Federal and State Governments (EXC- 294, BIOSS – Centre for
Biological Signaling). We thank J. Schmidt, D. Schächtele, and
J. Meßmer (University of Freiburg) for designing and constructing
the illumination boxes.
References
1. Hubbard MJ, Cohen P (1993) On target with
a new mechanism for the regulation of protein
phosphorylation. Trends Biochem Sci 18:172–
177. doi:10.1016/0968-0004(93)90109-Z
2. Tischer D, Weiner OD (2012) Illuminating
cell signalling with optogenetic tools. Nat Rev
Mol Cell Biol 15:551–558. doi:10.1038/
nrm3837
3. Beyer HM, Naumann S, Weber W, Radziwill G
(2015) Optogenetic control of signaling in
mammalian cells. Biotechnol J 10:273–283.
doi:10.1002/biot.201400077
4. Wend S, Wagner HJ, Müller K et al (2014)
Optogenetic control of protein kinase activity
in mammalian cells. ACS Synth Biol 3:280–
285. doi:10.1021/sb400090s
5. Chatelle CV, Hövermann D, Müller A et al
(2016) Optogenetically controlled RAF to
characterize BRAF and CRAF protein kinase
inhibitors. Sci Rep 6:23713. doi:10.1038/
srep23713
6. Katsura Y, Kubota H, Kunida K et al (2015)
An optogenetic system for interrogating the
temporal dynamics of Akt. Sci Rep 5:14589.
doi:10.1038/srep14589
7. Ahmad M, Cashmore AR (1993) HY4 gene of
A. thaliana encodes a protein with characteristics of a blue-light photoreceptor. Nature
366:162–166. doi:10.1038/366162a0
8. Cui Y, Choudhury SR, Irudayaraj J (2014)
Quantitative real-time kinetics of optogenetic
proteins CRY2 and CIB1/N using singleOptogenetic Control of Protein Kinases
13. The residual amount of culture medium after aspiration gains
increasing significance with decreasing surface area. Therefore,
washing the wells with PBS in case of small well format is
recommended (i.e., 48- /96-well format).
14. Light intensities of 5 μmol/(m
2
s) or lower are strongly recommended, since they sufficiently trigger the optogenetic System,
while showing negligible phototoxicity.
15. Illuminate the cell culture lab by safelight LEDs (i.e., wavelength longer than 500 nm). Measure the wavelength of LEDs
to ensure that they will not excite the blue light-responsive
CRY2/CIBN system.
16. After the centrifugation the almost clear pellet may be hard to
detect. Make sure not to transfer it, since it will lower the quality of the Western blot.
17. When boiling samples use Eppendorf Safe-Lock tubes. This
will prevent accidental opening of the tubes during boiling,
which leads to uneven protein amounts between samples.
18. Do not process too many samples at once. In case of the
CRY2/CIBN system, the light-induced interaction of the
fusion proteins is limited when working under safe light.
Acknowledgments
This work was supported by the Excellence Initiative of the German
Federal and State Governments (EXC- 294, BIOSS – Centre for
Biological Signaling). We thank J. Schmidt, D. Schächtele, and
J. Meßmer (University of Freiburg) for designing and constructing
the illumination boxes.
References
1. Hubbard MJ, Cohen P (1993) On target with
a new mechanism for the regulation of protein
phosphorylation. Trends Biochem Sci 18:172–
177. doi:10.1016/0968-0004(93)90109-Z
2. Tischer D, Weiner OD (2012) Illuminating
cell signalling with optogenetic tools. Nat Rev
Mol Cell Biol 15:551–558. doi:10.1038/
nrm3837
3. Beyer HM, Naumann S, Weber W, Radziwill G
(2015) Optogenetic control of signaling in
mammalian cells. Biotechnol J 10:273–283.
doi:10.1002/biot.201400077
4. Wend S, Wagner HJ, Müller K et al (2014)
Optogenetic control of protein kinase activity
in mammalian cells. ACS Synth Biol 3:280–
285. doi:10.1021/sb400090s
5. Chatelle CV, Hövermann D, Müller A et al
(2016) Optogenetically controlled RAF to
characterize BRAF and CRAF protein kinase
inhibitors. Sci Rep 6:23713. doi:10.1038/
srep23713
6. Katsura Y, Kubota H, Kunida K et al (2015)
An optogenetic system for interrogating the
temporal dynamics of Akt. Sci Rep 5:14589.
doi:10.1038/srep14589
7. Ahmad M, Cashmore AR (1993) HY4 gene of
A. thaliana encodes a protein with characteristics of a blue-light photoreceptor. Nature
366:162–166. doi:10.1038/366162a0
8. Cui Y, Choudhury SR, Irudayaraj J (2014)
Quantitative real-time kinetics of optogenetic
proteins CRY2 and CIB1/N using singleOptogenetic Control of Protein Kinases