Analytical gel filtration

Rahul Sharma; Michael Adams; Simonne Griffith-Jones; Tobias Sahr; Laura Gomez-Valero; Felix Weis; Michael Hons; Sarah Gharbi; Rayene Berkane; Alexandra Stolz; Carmen Buchrieser; Sagar Bhogaraju

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Analytical gel filtration

RS Rahul Sharma

MA Michael Adams

SG Simonne Griffith-Jones

TS Tobias Sahr

LG Laura Gomez-Valero

FW Felix Weis

MH Michael Hons

SG Sarah Gharbi

RB Rayene Berkane

AS Alexandra Stolz

CB Carmen Buchrieser

SB Sagar Bhogaraju

This method is extracted from research article: Nat Commun, Nov 2023

Structural basis for the toxicity of Legionella pneumophila effector SidH

DOI: 10.1038/s41467-023-42683-8

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To test the interaction between SidH^paris N-term and SidH^paris C-term, analytical gel filtrations were performed using 10 µM of both of these proteins. The sample was incubated on ice for 30 min before being injected into a pre-equilibrated (100 mM NaCl, 10 mM HEPES pH 7.5, 0.5 mM TCEP) Superdex SD200 3.2/300 size exclusion column (GE Life Sciences). The fractions were loaded on SDS-PAGE to visualize the protein bands. The resulting chromatograms were plotted using GraphPad Prism.

10 µM of apo SidH^paris N-term was incubated with 10 µM of in vitro transcribed tRNA^Phe (a kind help from Eva Kowalinski Lab) and 10 µM of E. coli purified EF-Tu in the presence of 1 mM GTP (Merck) and 2.5 mM MgCl₂ on ice for 30 min. The mix was loaded onto pre-equilibrated (100 mM NaCl, 10 mM Hepes pH 7.5, 2.5 mM MgCl₂) S200 3.2/300 size exclusion column.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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