Design and preparation of synthetic reference peptides for APP/Aβ TOMAHAQ proteomics, version 2
By onThis protocol describes the design and preparation of synthetic reference peptides for APP/Aβ TOMAHAQ proteomics.
A fluorescence-based in vitro scrambling assay for yeast MCP1 and human XK
By onVPS13 proteins are proposed to function at contact sites between organelles as bridges for lipids to move directionally and in bulk between organellar membranes. VPS13s are found to interact with integral membrane proteins, like MCP1 in yeast or XK in humans. We showed that MCP1 and XK scramble phospholipids in vitro. Here I describe the detailed procedure of purification, reconstitution, and scrambling assay for both MCP1 and XK.
In vitro GCase activity assay (total cell lysate)
By onGlucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide (GlcCer), a membrane glyco-sphingolipid, to ceramide and glucose. This assay detects GBA activity by using a fluorogenic substrate that reacts with cell lysates previously treated with or without CBE (GBA1 inhibitor). This protocol is part of a Collection of protocols (dx.doi.org/10.17504/protocols.io.8epv593dng1b/v1) for the paper "Glucocerebrosidase, a Parkinson´s disease-associated protein, is imported into mitochondria and regulates complex I assembly and function" (https://doi.org/10.21203/rs.3.rs-1521848/v1)
Induction of non-selective bulk autophagy
By onThis protocol describes how to induce bulk (non-selective) autophagy in HeLa cells through nutrient starvation.
Cell lysis and gel electrophoresis for protein analysis of HeLa cells
By onThe authors present multiple protocols used for biochemical analysis of protein expression and association. T
Immunoblotting analysis of samples from GolgiTAG (TMEM115-3HA) immunoprecipitation
By onAn immunoblotting method to assess efficient enrichment of Golgi proteins in Golgi immunoprecipitation products compared to whole cell lysates, and the purity of the immunoprecipitated Golgi by monitoring the expression of other organelles’ markers.
Native-PAGE analysis of VCP hexamer
By onThis protocol is associated with the following preprint, published on February 19th 2022: The AAA+ chaperone VCP disaggregates Tau fibrils and generates aggregate seeds Itika Saha, Patricia Yuste-Checa, Miguel Da Silva Padilha, Qiang Guo, Roman Körner, Hauke Holthusen, Victoria A. Trinkaus, Irina Dudanova, Rubén Fernández-Busnadiego, Wolfgang Baumeister, David W. Sanders, Saurabh Gautam, Marc I. Diamond, F. Ulrich Hartl, Mark S. Hipp bioRxiv 2022.02.18.481043; doi: https://doi.org/10.1101/2022.02.18.481043
Immunological detection of APP and proteins of the endolysosomal system
By onThis protocol describes the immunological detection of APP and proteins of the endolysosomal system.Here we present a general protocol for immunological detection by Western blotting of APP and proteins of the endolysosomal system, including EEA1, RAB5, PSEN1, LAMP1, LAMP2, TMEM192, and BACE1.
Sectioning of Mouse Brain by Cryostat
By onThis protocol describes how to use the cryostat to prepare and slice mouse brain sections for Immunohistochemistry.
The impact of MG149, a KAT8 inhibitor, on mitophagy: cell-based in vitro assays
By onProtocol outlines in vitro assays (TMRM, mitoKeima) for evaluating MG149 and KAT8 inhibition effects on mitophagy.
AAV Purification Protocol with Iodixanol gradient
By onProtocol used in the Kaplitt and Marongiu labs to purify AAVs.
Fluorescent Immunolabelling for Alpha-Synuclein in neuronal primary culture (Testing PFF toxicity)
By onThis protocol is designed to perform fluorescent immunolabelling on neuronal primary culture after PFF incubation. The labelling of phosphorylated alpha-synuclein is considered as a marker of the PFF toxicity and should be done to validate the PFFs.
Pole Test – mice
By onThis test assesses motor coordination in mice by assessing a mouse's ability to turn and descend on a vertical pole.
NHS-ester-protein-labeling
By onProtocol for labeling a purified protein with an NHS ester fluorescent dye.
Lysosome proteolysis analysis with DQ-BSA
By onProtocol to analyze lysosomal proteolysis in astrocytes generated from iPSCs.
Analysis of glycosphingolipids from human cerebrospinal fluid
By onThe method uses the fluorescent compound anthranilic acid (2-AA) to label oligosaccharides prior to analysis using normal-phase high-performance liquid chromatography. The labeling procedure is rapid, selective, and easy to perform.
GFP-YIPF3 Immunoprecipitation
By onThe protocol outlines the immunoprecipitation procedure for GFP-YIPF3 protein.
Gene editing of YIPF4, FIP200, and ATG7 in HEK293 cells
By onProtocol outlines creation of YIPF4, FIP200, and ATG7 knockout cell lines in HEK293 cells via CRISPR-Cas9 method.
