Protocol Archive

This protocol assumes that 2000 to 4000 movies of full-length LRRK2 embedded on vitreous ice are collected with an electron microscope equipped with a direct detector. It focuses primarily on the monomeric population of LRRK2 and has been leading to 3-4 Å resolution structures.

Protein purification protocol for full-length LRRK1 as done by Leschziner and Reck-Peterson Labs. Original protocol by Janice M. Reimer and Yu Xuan Lin.

This quick guide provides key minimal steps for preparing the Titan/SerialEM for the tomogram data collection on lamella or in vitro specimens with a K3 camera. paceTOMO routine is also included for a typical tomogram data collection session. Please note that this is not an exhaustive guide, but summarizes the order of key steps.

This protocol describes how to visualize alpha-synuclein in STC-1 cells by DAB immunohistochemistry. It also works for other antibodies (e.g., 5-HT, CCK, GLP).

Protocol used to generate stable Flp-In T-REx-HEK 293 cell lines expressing WT or mutant GCase (E326K or L444P) as a V5-FLAG-tagged protein using a tetracycline-inducible system.

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).

Protocol used for immunoprecipitation of HSP60 and LONP1 in HEK cells to show the interaction with V5-Flag-tagged WT-GCase.

Expansion microscopy is a technique to visualize biological structures with higher spatial resolution than traditional microscopy methods.

Protocol used to pull down V5-FLAG-Gcase interacting proteins in HEK cells.

The authors developed this protocol to identify protein-protein interactions between the enzyme glucocerebrosidase (GCase) and other proteins in human iPSC-derived Neural Precursor Cells.

iPSC generation and gene correction (CRISPR-CAS9) protocol.

Collection of protocols of Deleidi Lab used in the publication: "Glucocerebrosidase is imported into mitochondria and preserves complex I integrity and energy metabolism."

This protocol describes the immunofluorescence staining of cells.

The protocol works for many other antibodies and the only change necessary for most antibodies is determining the optimal dilution of the primary antibody and the best type of antigen-retrieval to use.

The authors describe the PLA protocol that is routinely used in the laboratory to detect nitrated alpha-synuclein and nitration of mitochondrial enzymes such as SOD2 and the mitochondrial complex 1 subunit NDUFB8.

Live-cell imaging is a technique to visualize dynamic cellular processes in living biological samples.

This was adapted from Cat. # EGLP-35K to optimize the seeding density of the assay based on the cell line used (Hutu 80 enteroendocrine cells RRID: CVCL_1301); moreover, cell line used and media conditions used to characterize GLP-1release. The lowest level of GLP-1 that can be detected by this assay is 2 pM (100ul plasma sample size).

Comparing two cultures of a similar passage number, cell density, time that the cells have been in culture, and time since the medium was changed as all this influences mitochondrial respiration has proven best. Culture cells until they are almost/just confluent. Cell should have been fed recently before use.

Mitochondria complex activity assays measure the activity levels of the different complexes of the mitochondrial electron transport chain (ETC).

The authors combined it with DAB-TH staining that works by using an antibody to detect the presence of TH, followed by a reaction with a substrate (DAB) that results in the formation of a brown-colored product at the site of the antigen-antibody interaction.