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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
Nuclear cytoplasmic fractionation
This protocol describes nuclear cytoplasmic fractionation.
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Surface protein biotinylation
This 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.
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Hippocampal Neuronal Culture
This protocol describes the procedure for hippocampal neuronal cultures from new – born mouse pups.
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Protocol for Image processing and analysis of VPS13D recruitment to mitochondria
This protocol details the image processing and analysis of VPS13D recruitment to mitochondria as it was performed in https://doi.org/10.1083/jcb.202010004. The first part details the analysis of acute optogenetic recruitment, and the second part of basal recruitment under different conditions.
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Optogenetic experiments with iLID system
This protocol details experiments with the iLID optogenetic system as performed to acutely recruit Miro to mitochondria in https://doi.org/10.1083/jcb.202010004.
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Protocol for hippocampal neuronal cultures
This protocol details the procedure for preparation of neuronal cultures from mice hippocampi as it was performed in https://doi.org/10.1083/jcb.202010004 but can also be used to prepare cultures of cortical neurons.
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A fluorescence-based in vitro scrambling assay for yeast MCP1 and human XK
Detailed procedure of purification, reconstitution, and scrambling assay for both MCP1 and XK.
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Cell culture on EM grids and fluorescence microscopy imaging
This 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.
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VPS13D DNA plasmid generation
This protocol describes the basic molecular cloning technique utilized for the generation of VPS13D constructs in VPS13D bridges the ER to mitochondria and peroxisomes via Miro. This protocol and the enzymes included in it are commercialized by Takara Bio.
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Crystallization, data collection and structure solution of PxP-VAB
Protocol lists the steps to obtain PxP(Mcp1ct)-VABct crystals, X-ray diffraction data acquisition and obtaining the structure solution using molecular replacement with a model generated by AlphaFold2.
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Purifying Vps13-VAB domain proteins
In this protocol, we have listed the steps to purify the Vps13 adaptor binding domain (or VAB) with or without PxP motif fusion peptide derived from adaptor proteins Mcp1, Ypt35 or Spo71.
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Plunge freezing
This protocol describes the procedure of plunge freezing for subsequent cryo-electron tomography.
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Cell culture, transfection, and imaging
This protocol describes general procedures for culturing HeLa cells, transient transfection, and imaging using an Andor Dragonfly spinning disk confocal system.
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Cell culture, transfection, and imaging
This protocol details the general preparation of cells for imaging and also for imaging experiments involving cellular hypotonic shock and cytosolic Ca2+ changes as they were performed in https://doi.org/10.1083/jcb.202010004.
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