Size-exclusion chromatography analysis of purified NagS.
<p>Analytical gel-filtration chromatography was performed on a Superdex 200 10/300 GL column (Cytiva) equilibrated with buffer containing 20 mM HEPES pH 7.5, 300 mM NaCl, 5% glycerol, and 1 mM DTT. NagS was analyzed at two protein concentrations (35 μM and 70 μM). Elution volumes (V<sub>...
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2025
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| 總結: | <p>Analytical gel-filtration chromatography was performed on a Superdex 200 10/300 GL column (Cytiva) equilibrated with buffer containing 20 mM HEPES pH 7.5, 300 mM NaCl, 5% glycerol, and 1 mM DTT. NagS was analyzed at two protein concentrations (35 μM and 70 μM). Elution volumes (V<sub>e</sub>) (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3003514#pbio.3003514.s021" target="_blank">S7 Table</a>) were compared to a molecular weight (Mw) calibration curve above generated using a Protein Standard Mix under identical buffer conditions. The calibration curve followed the equation V<sub>e</sub> = –1.525ln(Mw) + 20.659 (R² = 0.964). Note: NagS eluted at 14.45 ml, which corresponds to an estimated molecular weight of ~60.04 kDa according to the calibration curve equation. Given that the calculated molecular weight of a NagS-His₆ monomer is 28.29 kDa, these results indicate that NagS exists as a dimer in the tested buffer. The data underlying this Figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3003514#pbio.3003514.s022" target="_blank">S1 Data</a>.</p> <p>(TIF)</p> |
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