Purification of Histidine-Tagged Luciferase

Chemically competent C43 cells were used for expression of HTL [38 (link)]. HTL was purified as described previously [21 (link),39 (link),40 (link)] in TS₁₃₀G buffer (20 mM Tris–HCl^pH ≈ 8, 130 mM NaCl, 10% (v/v) Glycerol) for density gradient experiments and with Tris substitution for 50 mM HEPES^pH ≈ 8 in size exclusion experiments (HS₁₃₀G buffer: 50 mM HEPES^pH ≈ 8, 130 mM NaCl, 10% (v/v) Glycerol) [21 (link),39 (link),40 (link)]. Throughout later stages of purification both buffers were supplemented with 0.02% cardiolipin (CL) as described previously [23 (link)].

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Troman L., Alvira S., Daum B., Gold V.A, & Collinson I. (2023). Interaction of the periplasmic chaperone SurA with the inner membrane protein secretion (SEC) machinery. Biochemical Journal, 480(4), 283-296.

Publication 2023

Buffers Cardiolipin Cells Glycerol Nacl Tris

Corresponding Organization : University of Bristol

Other organizations : University of Exeter

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Variable analysis

independent variables

Expression of HTL in chemically competent C43 cells

dependent variables

Purification and characterization of HTL

control variables

TS130G buffer (20 mM Tris–HCl pH ≈ 8, 130 mM NaCl, 10% (v/v) Glycerol) for density gradient experiments
HS130G buffer (50 mM HEPES pH ≈ 8, 130 mM NaCl, 10% (v/v) Glycerol) for size exclusion experiments
0.02% cardiolipin (CL) supplementation in both buffers during later stages of purification

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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