Preparation To Minimize Buffer Mismatch in Isothermal Titration Calorimetry Experiments.
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.
Department of Biology, Texas A&M University , 3474 TAMU, College Station, Texas 77843-3474, United States.
Xuelin
X
Bian
Department of Biology, Texas A&M University , 3474 TAMU, College Station, Texas 77843-3474, United States.
Steve W
SW
Lockless
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002021:Buffers; D002122:Calcium Chloride / Q000737:chemistry; D002151:Calorimetry; D004492:Edetic Acid / Q000737:chemistry; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry; D011188:Potassium / Q000737:chemistry / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013302:Streptomyces / Q000378:metabolism
D001426:Bacterial Proteins; D002021:Buffers; D008024:Ligands; D008051:Lipid Bilayers; D015221:Potassium Channels; D011994:Recombinant Proteins; C107790:prokaryotic potassium channel; D004492:Edetic Acid; D002122:Calcium Chloride; D011188:Potassium