specifically to mycobacteria [36], and identifying in vivo lipid–
protein interactions in the mycomembrane [37]. Here, we provide
protocols for using trehalose- and TMM-mimicking probes to
metabolically label mycolate-containing cell envelope components
in live mycobacteria.
2 Materials
2.1 TrehaloseMimicking Probes
1. 5 mM fluorescein-modified trehalose (FITC-Tre): Dissolve
3.7 mg of FITC-Tre in 1 mL of DMSO, sterile filter through
DMSO-resistant filter (e.g., 0.22 or 0.45μm, nylon or PTFE).
Store at À20
C, protect from light.
2. 5 mM-N,N-dimethylamino-1,8-naphthalimide-modified trehalose (DMN-Tre): Dissolve 2.8 mg DMN-Tre in 1 mL of
DMSO, filter with DMSO-resistant filter. Store at À20
C,
protect from light.
3. 5 mM azide-modified trehalose (6-TreAz): Dissolve1.8 mg of
6-TreAz (Click Chemistry Tools) in 1 mL of ultrapure water.
Filter and store at À20
C.
2.2 TMM-Mimicking
Probes (See Note 3)
1. 5 mM fluorophore-modified TMM (O-FITC-TMM): Dissolve
4.5 mg in 1 mL DMSO, sterile filter through DMSO-resistant
filter, store at À20
C, and protect from light.
2. 5 mM trans-cyclooctene-modified TMM (O-TCO-TMM):
Dissolve 3.3 mg in 1 mL of DMSO, sterile filter through
DMSO-resistant filter, and store at À20
C.
3. 5 mM ester-linked 6-heptynoyl-modified TMM (O-AlkTMMC7): Dissolve 2.3 mg in 1 mL of ultrapure water, sterile filter,
and store at À20
C.
4. 5
mM
amide-linked
6-heptynoyl-modified
TMM
(N-AlkTMM-C7): Dissolve 2.2 mg in 1 mL of ultrapure
water, sterile filter, and store at À20
C.
5. 5 mM azide-modified TMM (O-AzTMM-C10): Dissolve
2.7 mg in 1 mL of ultrapure water, sterile filter, and store at
À20
C.
2.3 Bacterial Cell
Culture
1. Bacterial strains: Mycobacterium smegmatis mc
2 155 (ATCC
700084).
2. Middlebrook 7H9 medium: In a clean 2 L graduated beaker,
add 4.7 g of Middlebrook 7H9 broth base. Add 887 mL
ultrapure water and 10 mL of 50% glycerol in ultrapure water
(v/v). Stir to completely dissolve the broth base, heating if
necessary. Add 2.5 mL of 20% Tween 80 (v/v). Allow to cool
to room temperature and add 100 mL albumin-dextroseTrehalose Probe Labeling
389
protein interactions in the mycomembrane [37]. Here, we provide
protocols for using trehalose- and TMM-mimicking probes to
metabolically label mycolate-containing cell envelope components
in live mycobacteria.
2 Materials
2.1 TrehaloseMimicking Probes
1. 5 mM fluorescein-modified trehalose (FITC-Tre): Dissolve
3.7 mg of FITC-Tre in 1 mL of DMSO, sterile filter through
DMSO-resistant filter (e.g., 0.22 or 0.45μm, nylon or PTFE).
Store at À20
C, protect from light.
2. 5 mM-N,N-dimethylamino-1,8-naphthalimide-modified trehalose (DMN-Tre): Dissolve 2.8 mg DMN-Tre in 1 mL of
DMSO, filter with DMSO-resistant filter. Store at À20
C,
protect from light.
3. 5 mM azide-modified trehalose (6-TreAz): Dissolve1.8 mg of
6-TreAz (Click Chemistry Tools) in 1 mL of ultrapure water.
Filter and store at À20
C.
2.2 TMM-Mimicking
Probes (See Note 3)
1. 5 mM fluorophore-modified TMM (O-FITC-TMM): Dissolve
4.5 mg in 1 mL DMSO, sterile filter through DMSO-resistant
filter, store at À20
C, and protect from light.
2. 5 mM trans-cyclooctene-modified TMM (O-TCO-TMM):
Dissolve 3.3 mg in 1 mL of DMSO, sterile filter through
DMSO-resistant filter, and store at À20
C.
3. 5 mM ester-linked 6-heptynoyl-modified TMM (O-AlkTMMC7): Dissolve 2.3 mg in 1 mL of ultrapure water, sterile filter,
and store at À20
C.
4. 5
mM
amide-linked
6-heptynoyl-modified
TMM
(N-AlkTMM-C7): Dissolve 2.2 mg in 1 mL of ultrapure
water, sterile filter, and store at À20
C.
5. 5 mM azide-modified TMM (O-AzTMM-C10): Dissolve
2.7 mg in 1 mL of ultrapure water, sterile filter, and store at
À20
C.
2.3 Bacterial Cell
Culture
1. Bacterial strains: Mycobacterium smegmatis mc
2 155 (ATCC
700084).
2. Middlebrook 7H9 medium: In a clean 2 L graduated beaker,
add 4.7 g of Middlebrook 7H9 broth base. Add 887 mL
ultrapure water and 10 mL of 50% glycerol in ultrapure water
(v/v). Stir to completely dissolve the broth base, heating if
necessary. Add 2.5 mL of 20% Tween 80 (v/v). Allow to cool
to room temperature and add 100 mL albumin-dextroseTrehalose Probe Labeling
389