Surface protein biotinylation
By Michelle onThis protocol describes surface protein labeling with biotin using EZ-Link Sulfo-NHSLC-Biotin. This chemical reacts with primary amines such as lysine but does not permeate cell membranes because of the charge. Thus, it only biotinylates surface proteins.
Unilateral intranigral AAV alpha synuclein mouse model
By Michelle onThis protocol details methods for the unilateral intranigral AAV alpha-synuclein injection.
Western blotting for LRRK2 signalling in macrophages
By Michelle onThis protocol describes the immunoblotting for components of the LRRK2 signalling pathway (LRRK2, LRRK2 pS935 and phospho-Rabs) using Invitrogen NuPage SDS-PAGE reagents and the BioRad Turbo Blot transfer system.
Yeast cells live fluorescence imaging
By Michelle onProtocol for sample preparation and live fluorescence imaging of yeast cells.
CoxII degradation assay to assess mitophagy
By Blythe Lloyd onThis protocol details the procedure to assess mitophagy by analyzing COXII degradation via Western blotting.
Collection of protocols for paper: “Glucocerebrosidase, a Parkinson´s disease-associated protein, is imported into mitochondria and regulates complex I assembly and function”
By Blythe Lloyd onThis is a collection of protocols used in a recent pre-print by the Deleidi Lab, Team Schapira. You can access pre-print at https://doi.org/10.21203/rs.3.rs-1521848/v1
Cell line construction and maintenance for Lyso-IP and Endo-IP analysis of amyloid precursor protein processing
By Blythe Lloyd onLyso-IP is a method that allows for the isolation of lysosomes for proteomics and metabolomics (dx.doi.org/10.17504/protocols.io.bybjpskn; dx.doi.org/10.17504/protocols.io.bx9hpr36). We have developed an analogous approach for purification of early/sorting endosomes (Endo-IP).
3D-correlative FIB-milling and Cryo-ET of Autophagic structures in Yeast Cells
By Michelle onThis protocol describes how to plunge-freeze yeast on EM grids and how to target autophagic structures by combining cryo confocal fluorescence data to FIB-milling and tomogram acquisition.
Cell culture on EM grids and fluorescence microscopy imaging
By Blythe Lloyd onThis protocol describes the general procedure of seeding mammalian cells on EM grids and confocal fluorescence microscopy imaging of grids for subsequent cryo-tomography experiments, performed.
Binding of Rab29 to the LRRK2 Armadillo domain by Microscale Thermophoresis
By Blythe Lloyd onMicroscale thermophoresis (MST) is a powerful tool to measure the affinities of interactions between proteins. We present here our method for determining the binding of Rab29 GTPase to the LRRK2 (Leucine rich repeat kinase 2) N-terminal Armadillo domain.
Behavioral Testing – Open Field and Dyskinesia Scoring
By Blythe Lloyd onThis protocol describes behavioral testing to assess motor deficits in mice. It includes open field locomotor testing and scoring of levodopa-induced dyskinesia.
Assaying starvation-induced autophagy in HeLa cells
By Blythe Lloyd onA method for assaying starvation-induced autophagy in HeLa cells that have been transfected with Halo-tagged constructs. This protocol was specifically used to investigate the impact of various mutations in the protein WIPI2 on starvation-induced autophagy in cells.
Analysis of glycosphingolipids from human plasma
By Blythe Lloyd onThe original qualitative method was published in 2004 and measured the oligosaccharides selectively released from glycosphingolipids using a ceramide glycanase enzyme derived from the medicinal leech. This is an updated protocol with the focus on achieving sensitive and reproducible quantitation of glycosphingolipids from human plasma samples
Activation Induced Marker (AIM) Staining Protocol
By Blythe Lloyd onThis protocol details about activation induced marker staining.