ABSTRACT
Protein misfolding and aggregation is a characteristic of many neurodegenerative disorders, including Alzheimer's and Parkinson's disease. The oligomers generated during aggregation are likely involved in disease pathogenesis and present promising biomarker candidates. However, owing to their small size and low concentration, specific tools to quantify and characterize aggregates in complex biological samples are still lacking. Here, we present single-molecule two-color aggregate pulldown (STAPull), which overcomes this challenge by probing immobilized proteins using orthogonally labeled detection antibodies. By analyzing colocalized signals, we can eliminate monomeric protein and specifically quantify aggregated proteins. Using the aggregation-prone alpha-synuclein protein as a model, we demonstrate that this approach can specifically detect aggregates with a limit of detection of 5 picomolar. Furthermore, we show that STAPull can be used in a range of samples, including human biofluids. STAPull is applicable to protein aggregates from a variety of disorders and will aid in the identification of biomarkers that are crucial in the effort to diagnose these diseases.
Subject(s)
Parkinson Disease , Protein Aggregates , Humans , Parkinson Disease/metabolismABSTRACT
Metallothioneins (MTs) are small molecular weight stress response proteins that play a central role as reservoir of essential divalent heavy metal cations such as zinc and copper, and also can diminish the effects of toxic heavy metals such as mercury and cadmium. Historically, MT has been considered to be an intracellular protein with roles to play in the management of heavy metals, as a regulator of cellular redox potential, and as a buffer of free radicals. Our recent studies have highlighted immunomodulatory role of MT in inflammatory diseases and also in the progression of metastatic cell movement. Hence, manipulation and detection of MT is essential for its possible use as a diagnostic and in therapeutic interventions of chronic inflammation. This review describes procedures used to detect MT using techniques such as western immunoblot, competition ELISA, flow cytometry and immunohistochemistry. Additionally, it also describes the use of a colorimetric cell proliferation assay (CellTiter 96 AQueous One Solution/MTS) to study the proliferative effect of MT. © 2017 by John Wiley & Sons, Inc.
Subject(s)
Metallothionein/metabolism , Oxidative Stress , Animals , Blotting, Western , Cell Proliferation/physiology , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Immunohistochemistry , Metallothionein/chemistry , Metallothionein/physiology , Paraffin Embedding , Protein Conformation , Spleen/cytologyABSTRACT
The biotin-binding capacity in the wells of a streptavidin-coated PCR plate were quantified by means of a fluorescence intensity assay utilising biotin-labelled fluorescein and a colorimetric assay using biotin-labelled alkaline phosphatase. The biotin binding capacities were determined to be 59 and 58 pmol respectively.
