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Catalog
ASAP is committed to accelerating the pace of discovery and informing a path to a cure for Parkinson’s disease through collaboration, research-enabling resources, and data sharing. We’ve created this catalog to showcase the research outputs and tools developed by ASAP-funded programs.
Protocol
Generation of stable cell lines via retroviral or lentiviral transduction
This protocol details how to generate stable cell lines using a retrovirus system (can also be used for lentivirus systems) and includes the production of retroviruses encoding the transgene of interest in HEK293FT and the subsequent transduction of the cell line that is intended to stably express the protein of interest.
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Protocol
Binding of Rab29 to the LRRK2 Armadillo domain by Microscale Thermophoresis
Microscale 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.
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Staining of cells with GolgiTracker for Golgi flow cytometry analysis
Method that allows the staining of intact Golgi using GolgiTracker for subsequent flow cytometry analysis. The analysis can provide a quantitative indication of the level of enrichment of the organelle immunoprecipitationand can be used as a quality control check for the purity of the immunoprecipitated Golgi.
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Introducing GolgiTag to Cells and Immunoprecipitation of Golgi
A protocol for rapidly purifying Golgi across various cell lines and tissues without any modification. The Golgi purified using this method are highly enriched, intact, contaminant-free and, depending on solubilisation buffer, could be used for various downstream applications, such as proteomics and metabolomics.
Access the interview with Rotimi Fasimoye, PhD, about this protocol.
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Reconstitution of LRRK2 membrane recruitment onto planar lipid bilayers
A method to monitor the recruitment of purified LRRK2 kinase onto planar lipid bilayers containing lipid-anchored Rab10 protein using Total Internal Reflection Fluorescence (TIRF) Microscopy.
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CRISPR-Cas9 screen in NIH-3T3 cells to identify modulators of LRRK2 function
This protocol describes a pooled, CRISPR Cas9 screen to identify modulators of LRRK2 activity. The CRISPR-Cas9 based screen is carried out in mouse cells using a ready-to-use pooled guide RNA (gRNA) mouse library consisting of 78,637 gRNAs targeting 19,674 genes and an extra 1,000 control gRNAs.
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Immunofluorescence imaging of cells expressing GolgiTAG (TMEM115-3HA) or HA-tagged non-Golgi annotated proteins
This method that can be used to verify the correct localisation of HA-tagged Golgi proteins (like TMEM115-3HA, or GolgiTAG), by assessing their colocalisation with known Golgi markers such as GM130, GOLGIN97 and ACBD3 or can be used to assess if HA-tagged, non-Golgi annotated proteins transiently expressed in cells localise at the Golgi.
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Preparation and imaging of enriched Golgi from GolgiTAG-IP using Transmission Electron Microscopy
This protocol describes how Golgi, isolated from cells by GolgiTAG immunoprecipitation (IP) (as described in dx.doi.org/10.17504/protocols.io.6qpvrdjrogmk/v1), can be prepared and imaged using TEM. This protocol can also be used to image any organelles isolated using various organelle-IP protocols that are available.
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Untargeted lipidomics analysis for Golgi immunopurification (Golgi-IP)
This protocol provides details for analyzing GolgiIP lipidomics samples using liquid chromatography mass spectrometry (LC-MS) for nonpolar lipid profiling.
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Assay for Dual Rab GTPase binding to the LRRK2 Armadillo Domain
The LRRK2 Armadillo domain contains multiple Rab GTPase binding sites. To show that the sites can be occupied simultaneously, we use this assay. The idea is to immobilize Rab8A, bind Armadillo domain, and test if phosphoRab10 can bind to Rab8A-immobilized Armadillo domain.
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Assay for PhosphoRab activation of LRRK2 Kinase
MST kinase phosphorylates Rab proteins are at the same site as LRRK2 and have been used to phosphorylate Rab8A and Rab10 quantitatively. This protocol includes a method to produce phosphoRab8A protein and remove as much contaminating MST3 as possible, to enable use of the phosphoRab to test subsequent activation of LRRK2 kinase.
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Golgi immunopurification (Golgi-IP) for subcellular metabolite profiling
This protocol provides details for preparing Golgi-IP metabolomics samples.
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Untargeted metabolomics analysis for Golgi immunopurification (Golgi-IP)
This protocol provides details for analyzing GolgiIP metabolomics samples using liquid chromatography mass spectrometry (LC-MS) for polar metabolite profiling.
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Golgi immunopurification (Golgi-IP) for subcellular lipid profiling
This protocol provides details for preparing Golgi-IP lipidomics samples.
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