Flow cytometry technique detects translocating proteins
Detecting the translocation of proteins from one subcellular compartment to another is of interest to many researchers because translocation can reflect significant events in signal transduction in living cells. The protein-tyrosine kinase Syk offers an example. Syk is crucial to the survival, proliferation and differentiation of B lymphocytes, and the protein’s translocation is part of its role in signal transduction for each process.
Conventional techniques used for such detection are limited, though. Investigators have used subcellular fractionation/Western blotting or have imaged low numbers of cells; however, these techniques either do not offer the high throughput recommended for studying entire cell populations, or they provide only averaged information from the populations.
In a paper published in the Feb. 15 issue of Analytical Chemistry, researchers with Purdue University in West Lafayette, Ind., reported a technique they refer to as electroporative flow cytometry, which they used to address these shortcomings. With information acquired from single cells, they were able to distinguish populations in which translocation had occurred from those in which it had not — thus achieving high-throughput detection without resorting to averaged information.
Researchers have reported a technique they refer to as electroporative flow cytometry. The method enables high-throughput detection of protein translocation because the amount of material remaining in a cell after electroporation — in this case, the material is the protein Syk, tagged with enhanced GFP — correlates with whether or not translocation has occurred. Image courtesy of Chang Lu, Purdue University.
Electroporation is the result of a cell encountering an electrical field with a certain intensity: The electrical field induces the opening of pores in the cellular membrane, leading to the release of intracellular materials into the surrounding solution.
Using a homemade microfluidic device mounted on an Olympus inverted fluorescence microscope, the scientists showed that the amount of enhanced GFP-tagged Syk remaining in the cells after electroporation correlated with whether or not translocation of the protein had occurred. Furthermore, they demonstrated that by detecting translocation based on the release of intracellular kinase in individual cells, they could uncover characteristics of the entire cell population.
The researchers noted that the technique could contribute to kinase-related drug discovery as well as to tumor diagnosis and staging, since the translocations often are involved in oncogenesis and other similar disease processes.
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