The PMSF stock needs vigorous agitation for proper dissolution in a cold aqueous solvent. However, the next sonication
step helps to increase its solubilization.
12. The equipment used in this protocol was the Branson Ultrasonic Cell Disruptor SFX550 equipped with a 3 mm tip. Sonicators may vary notably in their performance and lysis
efficiency. So optimal sonication conditions have to be determined for every particular equipment. Use as a reference the
power settings routinely used for the sonication of E. coli cells.
Monitor the lysis by measuring the size of the pellet after
successive sonication steps (a microcentrifuge can be used).
In our experience, the pellet size reaches a plateau (almost
invariant pellet size) after a few sonication pulses.
13. Tools for the conversion of centrifugation settings among
Beckman rotors are available at https://www.beckman.com/
centrifuges/rotors/calculator. Note that increasing centrifugation times may reduce band resolution.
14. Electron microscopy observations and preliminary proteomics
and metabolomics analysis have shown the following main
features for each one of the isolated fractions. Fraction 1:
contains colorless plastoglobules, its protein content is
extremely low and is relatively rich in carotenoid precursors
(phytoene and phytofluene). Fraction 2: is enriched in plastoglobules and crystals. Has a low protein content and relatively
high levels of β-carotene. Fraction 3: contains plastoglobules
and crystals and has a low protein content. Is relatively enriched
in lycopene and β-carotene. Fraction 4: contains plastoglobulelike particles and other membranous components. Shows the
presence of lycopene and β-carotene. Fraction 5: Contains
membranous and vesicle-like structures. Shows the presence
of lycopene and β-carotene and is enriched in MGDG and
DGDG. Inner-membrane proteins have been identified. Fraction 6: it contains membranous structures and has a high
protein content; is enriched in carotenoid precursors (phytoene and phytofluene) and tocopherols; and contains relatively high levels of MGDG, DGDG, and phospholipids.
Outer-membrane proteins have been identified. Fraction 7: it
contains membranous components and vesicle-like structures
and has a relatively high protein content.
Acknowledgments
We acknowledge the financial support of AGAUR-Generalitat de
Catalunya (Grant 2017 SGR 710), the CERCA Programme of the
Generalitat de Catalunya and the Severo Ochoa Programme for
Centres of Excellence in R&D 2016–2019 to CRAG (SEV-2015196
Karel De Pourcq and Albert Boronat
step helps to increase its solubilization.
12. The equipment used in this protocol was the Branson Ultrasonic Cell Disruptor SFX550 equipped with a 3 mm tip. Sonicators may vary notably in their performance and lysis
efficiency. So optimal sonication conditions have to be determined for every particular equipment. Use as a reference the
power settings routinely used for the sonication of E. coli cells.
Monitor the lysis by measuring the size of the pellet after
successive sonication steps (a microcentrifuge can be used).
In our experience, the pellet size reaches a plateau (almost
invariant pellet size) after a few sonication pulses.
13. Tools for the conversion of centrifugation settings among
Beckman rotors are available at https://www.beckman.com/
centrifuges/rotors/calculator. Note that increasing centrifugation times may reduce band resolution.
14. Electron microscopy observations and preliminary proteomics
and metabolomics analysis have shown the following main
features for each one of the isolated fractions. Fraction 1:
contains colorless plastoglobules, its protein content is
extremely low and is relatively rich in carotenoid precursors
(phytoene and phytofluene). Fraction 2: is enriched in plastoglobules and crystals. Has a low protein content and relatively
high levels of β-carotene. Fraction 3: contains plastoglobules
and crystals and has a low protein content. Is relatively enriched
in lycopene and β-carotene. Fraction 4: contains plastoglobulelike particles and other membranous components. Shows the
presence of lycopene and β-carotene. Fraction 5: Contains
membranous and vesicle-like structures. Shows the presence
of lycopene and β-carotene and is enriched in MGDG and
DGDG. Inner-membrane proteins have been identified. Fraction 6: it contains membranous structures and has a high
protein content; is enriched in carotenoid precursors (phytoene and phytofluene) and tocopherols; and contains relatively high levels of MGDG, DGDG, and phospholipids.
Outer-membrane proteins have been identified. Fraction 7: it
contains membranous components and vesicle-like structures
and has a relatively high protein content.
Acknowledgments
We acknowledge the financial support of AGAUR-Generalitat de
Catalunya (Grant 2017 SGR 710), the CERCA Programme of the
Generalitat de Catalunya and the Severo Ochoa Programme for
Centres of Excellence in R&D 2016–2019 to CRAG (SEV-2015196
Karel De Pourcq and Albert Boronat