translocation and successfully used for biophysical and structural
analyses by electron cryo-microscopy and small-angle neutron scattering [14–16]. A scheme for the general workflow of holotranslocon production and parameters to be optimized is presented
in Fig. 1b.
2 Materials
Prepare all solutions using double-distilled water and analytical
grade reagents. Store all stock solutions at 4
C.
2.1 Expression
of HTL
1. pACEMBL HTL3 plasmid [15] (Fig.1a).
2. Chemically competent or electrocompetent E. coli BL21 Star
(DE3) cells (Thermo Fisher) or E. coli C43λ(DE3) cells [3] are
prepared according to published protocols [24, 25].
3. Prepare antibiotic stock solutions: 100 mg/mL ampicillin,
25 mg/mL chloramphenicol (dissolved in EtOH), and
50 mg/mL kanamycin.
4. Prepare LB medium: 10 g/L Bacto tryptone, 5 g/L yeast
extract, 10 g/L NaCl (verify pH 7.5, or adjust with NaOH),
and autoclave.
5. Prepare LB agar: Add 15 g/L Bacto agar to 1 L LB medium
and autoclave.
6. Incubator 37
C.
7. Shaker 37
C.
8. Spectrophotometer.
9. Baffled 5 L Erlenmeyer flasks.
10. Prepare Terrific Broth: 24 g/L yeast extract, 20 g/L Bacto
tryptone, 4 mL/L glycerol, 0.017 M KH 2 PO 4 , 0.072 M
K 2 HPO 4 , and autoclave.
11. Prepare stock solutions of 10% L-arabinose by dissolving 1 g Larabinose in 10 mL of water. Filter with 0.22 μm syringe filter
and store aliquots at 4
C. 1 M IPTG (isopropyl β-D-1-thiogalactopyranoside) is prepared by dissolving 2.38 g IPTG in
10 mL of water. Filter with a 0.22 μm syringe filter.
12. Centrifuge, e.g., Sorvall Lynx 6000 with F9 6x1000 Lex rotor
(Thermo Fisher).
2.2 Membrane
Preparation
1. French Press (Sim Aminco, Spectronic Instruments) or Cell
Disrupter/Microfluidizer (Microfluidics).
2. Centrifuge, e.g., Sorvall RC6 and SS-34 rotor (Sorvall).
3. Prepare 1 L HSGM buffer: 20 mM HEPES-KOH pH 8.0
(20 mL of 1 M stock solution), 130 mM NaCl (26 mL of
8
Burak V. Kabasakal et al.
analyses by electron cryo-microscopy and small-angle neutron scattering [14–16]. A scheme for the general workflow of holotranslocon production and parameters to be optimized is presented
in Fig. 1b.
2 Materials
Prepare all solutions using double-distilled water and analytical
grade reagents. Store all stock solutions at 4
C.
2.1 Expression
of HTL
1. pACEMBL HTL3 plasmid [15] (Fig.1a).
2. Chemically competent or electrocompetent E. coli BL21 Star
(DE3) cells (Thermo Fisher) or E. coli C43λ(DE3) cells [3] are
prepared according to published protocols [24, 25].
3. Prepare antibiotic stock solutions: 100 mg/mL ampicillin,
25 mg/mL chloramphenicol (dissolved in EtOH), and
50 mg/mL kanamycin.
4. Prepare LB medium: 10 g/L Bacto tryptone, 5 g/L yeast
extract, 10 g/L NaCl (verify pH 7.5, or adjust with NaOH),
and autoclave.
5. Prepare LB agar: Add 15 g/L Bacto agar to 1 L LB medium
and autoclave.
6. Incubator 37
C.
7. Shaker 37
C.
8. Spectrophotometer.
9. Baffled 5 L Erlenmeyer flasks.
10. Prepare Terrific Broth: 24 g/L yeast extract, 20 g/L Bacto
tryptone, 4 mL/L glycerol, 0.017 M KH 2 PO 4 , 0.072 M
K 2 HPO 4 , and autoclave.
11. Prepare stock solutions of 10% L-arabinose by dissolving 1 g Larabinose in 10 mL of water. Filter with 0.22 μm syringe filter
and store aliquots at 4
C. 1 M IPTG (isopropyl β-D-1-thiogalactopyranoside) is prepared by dissolving 2.38 g IPTG in
10 mL of water. Filter with a 0.22 μm syringe filter.
12. Centrifuge, e.g., Sorvall Lynx 6000 with F9 6x1000 Lex rotor
(Thermo Fisher).
2.2 Membrane
Preparation
1. French Press (Sim Aminco, Spectronic Instruments) or Cell
Disrupter/Microfluidizer (Microfluidics).
2. Centrifuge, e.g., Sorvall RC6 and SS-34 rotor (Sorvall).
3. Prepare 1 L HSGM buffer: 20 mM HEPES-KOH pH 8.0
(20 mL of 1 M stock solution), 130 mM NaCl (26 mL of
8
Burak V. Kabasakal et al.