Filter Retardation Assay for Detecting and Quantifying Polyglutamine Aggregates Using Caenorhabditis elegans Lysates

Abstract

Protein aggregation is a hallmark of several neurodegenerative diseases and is associated with impaired protein homeostasis. This imbalance is caused by the loss of the protein's native conformation, which ultimately results in its aggregation or abnormal localization within the cell. Using a C. elegans model of polyglutamine diseases, we describe in detail the filter retardation assay, a method that captures protein aggregates in a cellulose acetate membrane and allows its detection and quantification by immunoblotting.

The Proteasome Activators Blm10/PA200 Enhance the Proteasomal Degradation of N-Terminal Huntingtin

“…The filter trap assay was performed to analyze SDS-insoluble aggregates according to the published literature [ 45 , 46 ]. After 48 h of transient transfection with pHM6-HA-Q23 and pHM6-HA-Q74, cells were rinsed with 1X PBS and lysed with lysis buffer (40 mM HEPES, pH 7.5, 50 mM KCl, 1% ( v / v ) Triton X-100, 2 mM DTT, 5 mM EDTA) supplemented with 1X cOmplete™, Mini, EDTA-free protease inhibitor cocktail.…”

Exploiting flow cytometry for the unbiased quantification of protein inclusions in Caenorhabditis elegans

“…For experiments assaying worms at different ages, NGM plates containing 50 μM 5-fluorodeoxyuridine (FUDR) were used instead of standard NGM plates to inhibit egg-laying, as described previously (Sutphin & Kaeberlein, 2009). In these experiments, worms were transferred to fresh FUDR-containing plates whenever OP50 bacteria were close to being depleted (approximately every 3 days).…”

“…In addition, these worms have a short reproductive cycle and are easy to manipulate genetically. Consequently, they have been investigated extensively in forward and reverse genetic studies to identify genes that modulate biochemical processes including those involved in protein aggregation and neurodegeneration (Kraemer et al, 2006;Nollen et al, 2004;van Ham et al, 2008). They are also highly amenable to screens of pharmacological compounds and are often used as an initial model system for potential therapeutics before conducting rodent-based studies (Chen et al, 2015;Ikenaka et al, 2019).…”

“…A fundamental component of studying the aggregation of proteins in cells and organisms, and identifying modulators of this process, is the capacity to quantify the number and type of inclusions that are formed. The quantification of aggregation in C. elegans often requires laborious manual counting of inclusions from images generated through fluorescence microscopy, or other biochemical methods, such as fractionation of detergent soluble and insoluble fractions or filter trap assays, both of which rely on quantifying the amount of insoluble protein in the lysate (Sin et al, 2018; Walther et al, 2017). Recently, there have been several advances in automated imaging methods in an attempt to increase the throughput of microscopy‐based experiments (Cornaglia et al, 2016; Gosai et al, 2010; Mondal et al, 2016).…”

Exploiting flow cytometry for the unbiased quantification of protein inclusions inCaenorhabditis elegans

“…A fundamental component of studying the aggregation of proteins in cells and organisms, and identifying modulators of this process, is the capacity to quantify the number and type of inclusions that are formed. The quantification of aggregation in C. elegans often requires laborious manual counting of inclusions from images generated through fluorescence microscopy, or other biochemical methods, such as immunoblotting or filter trap assays, which rely on quantifying the amount of insoluble protein in the lysate (Sin et al 2018; Walther et al 2017). Recently, there have been several advances in automated imaging methods in an attempt to increase the throughput of microscopy-based experiments (Cornaglia et al 2016; Gosai et al 2010; Mondal et al 2016).…”