Purification and Analysis of Protein

UV-Visible spectra of all samples were measured using a U-3900 spectrophotometer (Hitachi). During Nickel-Nitrilotriacetic acid (Ni-NTA) affinity chromatography, fractions were analysed by UV-Visible spectroscopy to determine the A410/A280 ratio (the so-called Rz value). Fractions with an Rz value ³ of 0.5 were collected and used for subsequent purification. After size exclusion chromatography, fractions with an Rz value ³ of 0.7 were concentrated and subsequently used for cryoEM analysis.

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Gopalasingam C.C., Egami H., Shigematsu H., Sakaue M., Fukumoto K., Gerle C., Yamamoto M., Shiro Y., Muramoto K., & Tosha T. (2024). Monomer-dimer structural comparison in quinol-dependent nitric oxide reductase reveals a functional basis for superior enzymatic activity in the dimer.

Publication 2024

Corresponding Organization : SPring-8

Other organizations : Japan Synchrotron Radiation Research Institute

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Variable analysis

independent variables

Ni-NTA affinity chromatography
Size exclusion chromatography

dependent variables

A410/A280 ratio (Rz value)
Fractions collected for subsequent purification

control variables

U-3900 spectrophotometer (Hitachi) used for UV-Visible spectra measurements

controls

Fractions with an Rz value ≥ 0.5 were collected and used for subsequent purification
Fractions with an Rz value ≥ 0.7 were concentrated and subsequently used for cryoEM analysis

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