This website uses cookies so that we can provide you with the best user experience possible. Cookie information is stored in your browser and performs functions such as recognising you when you return to our website and helping our team to understand which sections of the website you find most interesting and useful.
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
Microscopy-based bead protein-protein interaction assay
This protocol describes how to perform microscopy-based bead protein-protein interaction assay with GST- or mCherry-tagged proteins as baits and fluorescently-tagged proteins as preys. The protocol requires to have purified proteins and allows to monitor protein-protein interaction in an equilibrium state. The fluorescent signal can be quantified.
Teams
Themes
Protocol
HeLa culture, transfection, and labeling of Halo-fusion proteins
The authors developed a protocol to investigate the role of the NEMO in Parkin-dependent mitochondrial clearance. Halo-fusion constructs, including NEMO and OPTN used in the study, allowed the authors to visualize the exogenously expressed proteins conjugated to chemical ligands in a variety of colors. This and the accompanying protocols were critical to the characterization of NEMO’s involvement in mitophagy.
Teams
Themes
Protocol
Expression and purification of human NEMO (GST-GFP-NEMO)
This protocol describes how to express and purify human NEMO (IKK-γ) tagged N-terminally with GST and EGFP. The expression is performed with E. coli Rosetta pLysS cells. The protein is purified via a GST batch purification and gel filtration (SEC).
Teams
Themes
Protocol
Fixation and imaging of HeLa cells after mitochondrial depolarization
Ectopic expression of p62/SQSTM1 often induces puncta formation and poor cell health due to p62’s proclivity to multimerize and form filaments. We noted modifying fixation protocol to visualize various endogenous ATG8 proteins, which are difficult to over-express in our system. Fixation techniques are a critical complement to studies in live cells.
Teams
Themes
Protocol
Fixing hippo neurons to assess endogenous NEMO during oxidative stress
This protocol details how to assess endogenous NEMO during oxidative stress in fixed hippocampal rat neurons. Mitochondrial damage was induced by Antimycin A, endogenous NEMO was visualized with the commercially available anti-NEMO primary antibody (abcam), and mitochondria were visualized by a mito-targeted construct, Mito-SNAP.
Teams
Themes
Protocol
HEK293 cell culture for co-immunoprecipitation experiments
The authors describe HEK293 cell culture for the purpose of co-immunoprecipitation experiments.
Teams
Themes
Protocol
Image processing to investigate NEMO recruitment and involvement in mitophagy and inflammatory signaling
The authors approached image analysis in a multitude of creative, effective ways, and importantly the authors maintained consistency of analysis within experiments in order to present the results with integrity and reproducibility.
Teams
Themes
Protocol
Immunostaining of iPSC-derived neurons for quantification of synaptic proteins
Here, we fix, permeabilize, and stain human iPSC-derived neurons for the purpose of observing and quantifying somal proteins of interest. For preceding culture of neurons, see “Protocol: Culture and transfection of iPSC-derived neurons for live-imaging of axonal cargoes.”
Teams
Themes
Protocol
Live imaging and analysis to investigate phase separation properties of NEMO during mitophagy
Three standards of the field for determining phase separation are: (A) particles undergo fission and fusion like droplets of water, (B) individual puncta recover fluorescence after photobleaching, and (C) particles dissipate upon exposure to 1,6-Hexanediol. This protocol describes the authors’ approach to investigating standards B and C.
Teams
Themes
Protocol
Live imaging to investigate mitophagy kinetics and NEMO recruitment in HeLa-M cells
The live imaging of NEMO occupancy on damaged mitochondria was a necessary complement to the authors’ parallel fixation studies. Their reporting of these results would not have been possible without real-time, live cell imaging.
Teams
Themes
Protocol
Live-cell imaging for synaptic vesicle precursors in human iNeuron axons
The authors describe procedure and equipment used for live-imaging of synaptic vesicle precursors. This was performed using DIV21 human iPSC-derived excitatory glutamatergic neurons. Equipment and software used varied based on scheduled upgrades to microscopy equipment during the course of this study.
Teams
Themes
Protocol
In vitro kinase assay: Phosphorylation of PI3Kc1 by TBK1and ULK1
This protocol describes in vitro kinase assay, in which PI3Kc is tested for phosphorylation by TBK1 and ULK1 complex either by western blotting (S29 on ATG14L subunit) or mass spectrometry analysis.
Teams
Themes
Protocol
SINTBAD-GFP: Expression and purification
This protocol describes how to express and purify human SINTBAD tagged C-terminally with eGFP.
Teams
Themes
Protocol
NAP1-mCherry
This protocol describes how to express and purify human NAP1 tagged C-terminally with mCherry.
Teams
Themes