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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
Protocol Collection: Combinatorial selective ER-phagy remodels the ER during neurogenesis
This molecular inventory of ER proteome remodeling and versatile genetic toolkit provides a quantitative framework for understanding contributions of individual ER-phagy receptors for reshaping ER during cell state transitions.
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Clusterin cellular uptake assay
This protocol details how to efficiently monitor Clusterin and Clusterin/Substrate uptake in different cell types like HEK293T, iNeurons, and iMicroglia.
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N_terminal protein labeling
This protocol details how to efficiently label a protein at the N-terminus using Clusterin protein as example.
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Formation and isolation of Clu phospholipid particles
This protocol details how to efficiently make in vitro and isolate Clu-phospholipid particles using purified Clusterin from HEK293E cells (dx.doi.org/10.17504/protocols.io.bvvkn64w) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC).
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Very low-density lipoprotein receptor (VLDLR)-C-tag purification from HEK293E cells
This protocol details how to purify recombinant very low-density lipoprotein receptor (VLDLR)-C-tag protein from HEK293E cells.
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Lentivirus preparation for neuronal transdifferentiation
This is a protocol for the preparation of Lentiviruses.
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Neuronal transdifferentiation from human primary adult fibroblasts
This is a protocol for the direct conversion of human primary fibroblasts into neurons using a combination of transcription factor- and small molecule-based approach. The majority of converted neurons showing characteristics of cortical glutamatergic neurons.
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Gene editing of YIPF4 in hESCs V3
This protocol describes the creation of YIPF4 knockout cell lines in H9 hESC cells using CRISPR-Cas9.
Associated with preprint:
https://doi.org/10.1101/2022.12.06.519342
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Cytokine profiling analysis on conditioned medium of human neurons using Luminex multiplex assay
This protocol is used to identify secreted inflammatory factors by cytokine profiling of the conditioned medium from human transdifferentiated neurons of healthy donors and AD patients.
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Immunohistochemistry on free-floating and paraffin-embedded tissue sections
This protocol is used for free-floating frozen (30-50 microns) and paraffin-embedded (10 microns) tissue sections.
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Cloning, protein expression, and purification of 20S CPs and assembly intermediates
This protocol details methods for cloning, expression, and purification of 20S CPs, and assembly intermediates for biochemical and structural analysis.
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Structural analysis of 20S CPs and assembly intermediates by electron cryo-microscopy
This protocol details methods for structural determination by transmission electron cryo-microscopy of 20S CPs and assembly intermediates.
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Neural differentiation on EM grids – iNeurons sample preparation for cryo-ET and CLEM
A protocol for differentiating AAVS1-TRE3G-NGN2 hESCs to iNeurons directly on EM grids for cryo-ET and cryo-CLEM. The protocol includes: (1) EM grids coating with Matrigel or Poly-Ornithine and Laminin; (2) differentiation and seeding of iNeurons on EM grids; (3) lentiviral transduction for transient expression of fluorescently-labeled proteins; and (4) plunge freezing. Watch an interview about this protocol with Cristina Capitanio, MSc.
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