A Flexible, Robust Microbead-based Assay for Quantification and Normalization of Target Protein Concentrations
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Crowley, Alexandra
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Abstract
There are many methods for quantifying the concentration of a specific (target) protein in a sample, but current techniques are technically challenging or do not easily lend themselves to normalizing measured protein concentrations against the total amount of protein in the sample. Here, we describe a microbead-based assay for quantifying specific protein concentration(s) that is high-throughput, inexpensive, relatively simple to carry out, and which intrinsically incorporates normalization against the total protein content in each sample. This assay, which we term the FRANC assay, exploits high affinity biotin-streptavidin binding to couple sample proteins to streptavidin-labeled magnetic microbeads. Proteins so attached are then probed with one or more antibodies, followed by labeling of all proteins on the microbead with a fluorescent dye, and flow cytometry-based analysis. The FRANC assay demonstrates detection limits for target proteins in the single femtogram range, with an operating range up to as much as 10 nanograms of target protein. When quantifying total protein amount, the variation between different protein samples was similar to that of the bicinchoninic acid (BCA) assay. Finally, normalization of target protein concentrations resulted in at least an 80% reduction in variability as compared to non-normalized measurements. We conclude that the FRANC assay offers attractive advantages over current methods of quantifying specific protein(s) in complex samples.
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2019-05
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Undergraduate Thesis