Question

3 a) Which of the following is NOT a correct application of a technique useful for studying cell signaling?

pull-down assays using binding domains that recognize only activated GTP-binding proteins to measure activity in the presence or absence of exogenous factors

affinity chromatography used to isolate cell surface receptors

immunoprecipitation of protein kinases and in vitro phosphorylation assays

FRET to monitor binding of a paracrine ligand to a population of cells

3 B) Which of the following is NOT a correct application of a technique useful for studying cell signaling?

pull-down assays using binding domains that recognize only activated GTP-binding proteins to measure activity in the presence or absence of exogenous factors

affinity chromatography used to isolate cell surface receptors

immunoprecipitation of protein kinases and in vitro phosphorylation assays

FRET to monitor binding of a paracrine ligand to a population of cells

Answer 1

FRET to monitor binding of a paracrine ligand to a population of cells.

FRET measurements make use of two or more fluorescent labels, or proteins, each of which may present different sensitivities for changes in environmental factors (pH, ionic concentrations, oxidation, temperature, and refractive index). Hence, FRET measurements may be skewed by agonist-induced or unappreciated changes in local environment. Thus it is much more suitable for monitoring intracellular protein-ligand binding instead extracellular interactions.