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Anal Chem. 2016 May 17;88(10):5549-53. doi: 10.1021/acs.analchem.6b01319. Epub 2016 May 5.

Preparation To Minimize Buffer Mismatch in Isothermal Titration Calorimetry Experiments.

Author information

1
Department of Biology, Texas A&M University , 3474 TAMU, College Station, Texas 77843-3474, United States.

Abstract

There is a growing need to study ligand binding to proteins in native or complex solution using isothermal titration calorimetry (ITC). For example, it is desirable to measure ligand binding to membrane proteins in more native lipid-like environments such as bicelles, where ligands can access both sides of the membrane in a homogeneous environment. A critical step to obtain high signal-to-noise is matching the reaction chamber solution to the ligand solution, typically through a final dialysis or gel filtration step. However, to obtain reproducible bicelles, the lipid concentrations must be carefully controlled which eliminates the use of dialysis that can disrupt these parameters. Here, we report and validate a rapid preparation ITC (RP-ITC) approach to measure ligand binding without the need for a dialysis step. This general approach is used to quantify ion binding to a K(+) channel embedded in bicelles and can be applied to complex, less defined systems.

PMID:
27092566
DOI:
10.1021/acs.analchem.6b01319
[Indexed for MEDLINE]

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