title: Quantitation of secreted proteins using mCherry fusion constructs and a fluorescent microplate reader.
abstract: Traditional assays for secreted proteins include methods such as Western blot and enzyme-linked immunosorbent assay (ELISA) detection of the protein in the cell culture medium. We describe a method for the detection of a secreted protein based on fluorescent measurement of an mCherry fusion reporter. This microplate reader-based mCherry fluorescence detection method has a wide dynamic range of 4.5 orders of magnitude and a sensitivity that allows detection of 1 to 2fmol fusion protein. Comparison with the Western blot detection method indicated greater linearity, wider dynamic range, and a similar lower detection threshold for the microplate-based fluorescent detection assay of secreted fusion proteins. An mCherry fusion protein of matrix metalloproteinase-9 (MMP-9), a secreted glycoprotein, was created and expressed by transfection of human embryonic kidney (HEK) 293 cells. The cell culture medium was assayed for the presence of the fluorescent signal up to 32 h after transfection. The secreted MMP-9-mCherry fusion protein was detected 6h after transfection with a linear increase in signal intensity over time. Treatment with chloroquine, a drug known to inhibit the secretion of many proteins, abolished the MMP-9-mCherry secretion, demonstrating the utility of this method in a biological experiment.
authors.affiliation: Translational Cardiovascular Science Training Program, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA; Department of Anesthesiology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA.
authors.forename: Tyler
authors.initials: T
authors.lastname: Duellman
authors.affiliation: Department of Anesthesiology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA.
authors.forename: John
authors.initials: J
authors.lastname: Burnett
authors.affiliation: Translational Cardiovascular Science Training Program, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA; Department of Anesthesiology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA. Electronic address: jyang75@wisc.edu.
authors.forename: Jay
authors.initials: J
authors.lastname: Yang
mesh_terms: D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D020780:Matrix Metalloproteinase 9 / Q000032:analysis / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; D020780:Matrix Metalloproteinase 9
keywords: Fluorescence; MMP-9; Microplate; Secretion; mCherry