Virginia Polytechnic Institute and State University (Virginia Tech; Blacksburg, VA) researchers have developed a flow cytometry-based technique that detects the subcellular location of a protein, which could allow the scientific and technological communities a simple and improved method for studying effectiveness of therapies for disease, including cancer.
Virginia Tech chemical engineer Chang Lu and his colleagues have used a National Science Foundation grant to develop their technique, as they recognize that "simple and accessible detection methods that can rapidly screen a large cell population with the resolution of a single cell inside that population has been seriously lacking," he explains.
If a protein is not located in the right subcellular compartment of a cell, "the result can be diseases ranging from metabolic disorders to cancers," Lu explains. "Modulation of protein transport inside a cell is practiced as an important therapeutic approach for cancer treatment. The subcellular location of a target protein can also serve as a useful read-out for high-content screening of cancer drugs."
|Virginia Tech chemical engineer Chang Lu and his graduate student Zhenning Cao developed a unique process that could lead to better drug development. (Image courtesy of Virginia Tech)|
In the human body, proteins move between distinct compartments inside cells, including the plasma membrane, the nucleus, and other membrane-enclosed areas. This movement can be a prerequisite for proteins to carry out their intended functions. These functions might include gene transcription and other molecular regulations.
One current evaluation method of protein movement, fluorescence microscopy, can only analyze a limited number of cells, says Lu. And data collected by a second existing assay called subcellular fractionation only reflects the average properties of the cell populations "without revealing the heterogeneity that is often present among seemingly identical cells," Lu and other research team members say in their paper published in the Royal Society of Chemistry journal Chemical Science.
Lu’s team had made some progress in screening cell populations in the past using an electroporation-based technique, but it did not allow the examination of native proteins and primary cells isolated from animals and from patients. So, their new work uses a method that "incorporates selective chemical release of cytosolic proteins with a standard procedure for fluorescent labeling of the protein to detect the subcellular location of a native protein," Lu said. This simple and unique tweak to the conventional cell staining process allowed them to accurately define the subcellular location of the protein by measuring the amount of the residual protein after release. Using a flow cytometer, the speed of such measurement could reach 10,000 to 100,000 cells/s.
A key ingredient to their process is the use of saponin, a class of amphipathic glycosides. It dissolves cholesterol and permeates the plasma membrane to allow protein release. “Gentle treatment by saponin shows minimal effects on the state of the cell,” Lu adds.
To read the Chemical Science paper, please visit http://dx.doi.org/10.1039/C4SC00578C.
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