equivalent to OD 260 ¼ 1 U. Changing the gradient volume
will require appropriate change in rotors and tubes. For example, SW41 and SW55 rotors would be required to centrifuge
15 and 5 mL gradients, respectively.
12. If undisturbed, samples may be stored under this condition for
a few hours—an unavoidable situation when multiple samples
need to be fractionated.
13. For dilute samples of ribosomes, optical filter of 254 nm is
recommended.
14. See Fig. 2 for a typical profile of density gradient.
15. Antibodies exclusively recognizing C+ ribosomes, although
unavailable at the time of writing this protocol, can in principle
be included in the immunoblot analysis to determine the level
of cross contamination of C+ ribosomes in CÀ and hibernating
CÀ ribosomes.
16. Amounts of ribosomes and titer of Mpy antibodies must be
calibrated to capture the linear dynamic range of the detected
signal. Background Mpy signal in ribosome preparations from
high-zinc cultures often become prominent if more ribosomes
are loaded, or higher amounts of antibodies are used (Fig. 3).
Acknowledgments
Support from Tissue Culture and Media core facility of the Wadsworth Center in making the buffer stocks is acknowledged. Authors
also acknowledge funding support from the National Institute of
Health (AI132422 and AI144474 to A.K.O. and GM061576 to R.
K.A.) and New York State Department of Health in carrying out
this work.
References
1. Wilson DN, Nierhaus KH (2007) The weird
and wonderful world of bacterial ribosome regulation. Crit Rev Biochem Mol Biol 42
(3):187–219.
https://doi.org/10.1080/
10409230701360843
2. Wada A, Yamazaki Y, Fujita N, Ishihama A
(1990) Structure and probable genetic location
of a “ribosome modulation factor” associated
with 100S ribosomes in stationary-phase
Escherichia coli cells. Proc Natl Acad Sci U S
A 87(7):2657–2661. https://doi.org/10.
1073/pnas.87.7.2657
3. Maki Y, Yoshida H, Wada A (2000) Two proteins, YfiA and YhbH, associated with resting
ribosomes in stationary phase Escherichia coli.
Genes Cells 5(12):965–974
4. Ueta M, Wada C, Wada A (2010) Formation of
100S ribosomes in Staphylococcus aureus by
the hibernation promoting factor homolog
SaHPF. Genes Cells 15(1):43–58. https://
doi.org/10.1111/j.1365-2443.2009.
01364.x
5. Ueta M, Wada C, Daifuku T, Sako Y, Bessho Y,
Kitamura A, Ohniwa RL, Morikawa K,
Yoshida H, Kato T, Miyata T, Namba K,
Wada A (2013) Conservation of two distinct
types of 100S ribosome in bacteria. Genes Cells
18(7):554–574. https://doi.org/10.1111/
gtc.12057
6. Ueta M, Yoshida H, Wada C, Baba T, Mori H,
Wada A (2005) Ribosome binding proteins
YhbH and YfiA have opposite functions during
164
Yunlong Li et al.
will require appropriate change in rotors and tubes. For example, SW41 and SW55 rotors would be required to centrifuge
15 and 5 mL gradients, respectively.
12. If undisturbed, samples may be stored under this condition for
a few hours—an unavoidable situation when multiple samples
need to be fractionated.
13. For dilute samples of ribosomes, optical filter of 254 nm is
recommended.
14. See Fig. 2 for a typical profile of density gradient.
15. Antibodies exclusively recognizing C+ ribosomes, although
unavailable at the time of writing this protocol, can in principle
be included in the immunoblot analysis to determine the level
of cross contamination of C+ ribosomes in CÀ and hibernating
CÀ ribosomes.
16. Amounts of ribosomes and titer of Mpy antibodies must be
calibrated to capture the linear dynamic range of the detected
signal. Background Mpy signal in ribosome preparations from
high-zinc cultures often become prominent if more ribosomes
are loaded, or higher amounts of antibodies are used (Fig. 3).
Acknowledgments
Support from Tissue Culture and Media core facility of the Wadsworth Center in making the buffer stocks is acknowledged. Authors
also acknowledge funding support from the National Institute of
Health (AI132422 and AI144474 to A.K.O. and GM061576 to R.
K.A.) and New York State Department of Health in carrying out
this work.
References
1. Wilson DN, Nierhaus KH (2007) The weird
and wonderful world of bacterial ribosome regulation. Crit Rev Biochem Mol Biol 42
(3):187–219.
https://doi.org/10.1080/
10409230701360843
2. Wada A, Yamazaki Y, Fujita N, Ishihama A
(1990) Structure and probable genetic location
of a “ribosome modulation factor” associated
with 100S ribosomes in stationary-phase
Escherichia coli cells. Proc Natl Acad Sci U S
A 87(7):2657–2661. https://doi.org/10.
1073/pnas.87.7.2657
3. Maki Y, Yoshida H, Wada A (2000) Two proteins, YfiA and YhbH, associated with resting
ribosomes in stationary phase Escherichia coli.
Genes Cells 5(12):965–974
4. Ueta M, Wada C, Wada A (2010) Formation of
100S ribosomes in Staphylococcus aureus by
the hibernation promoting factor homolog
SaHPF. Genes Cells 15(1):43–58. https://
doi.org/10.1111/j.1365-2443.2009.
01364.x
5. Ueta M, Wada C, Daifuku T, Sako Y, Bessho Y,
Kitamura A, Ohniwa RL, Morikawa K,
Yoshida H, Kato T, Miyata T, Namba K,
Wada A (2013) Conservation of two distinct
types of 100S ribosome in bacteria. Genes Cells
18(7):554–574. https://doi.org/10.1111/
gtc.12057
6. Ueta M, Yoshida H, Wada C, Baba T, Mori H,
Wada A (2005) Ribosome binding proteins
YhbH and YfiA have opposite functions during
164
Yunlong Li et al.