Introducing GolgiTag to Cells and Immunoprecipitation of Golgi
By onProtocol for generating GolgiTag cells and immunoprecipitation of Golgi for subsequent -omics analyses.
Axonal and somatodendritic proteomes of dopamine neurons in the mouse brain
By onDopamine (DA) neurons modulate neural circuits and behaviors via dopamine release from expansive, long range axonal projections. The elaborate cytoarchitecture of DA neurons is embedded within complex brain tissues, making it difficult to access the DA neuronal proteome using conventional methods. Here, we demonstrate APEX2 proximity labeling within genetically targeted neurons in the mouse brain, enabling subcellular proteomics with cell type-specificity. By combining APEX2 biotinylation with mass spectrometry, we mapped the somatodendritic and axonal proteomes of DA neurons. Our dataset reveals the proteomic architecture underlying axonal transport, dopamine transmission, and axonal metabolism in DA neurons. We find a significant enrichment of proteins encoded by Parkinson’s disease-linked genes in dopaminergic axons, including proteins with previously undescribed axonal localization. Our proteomic datasets comprise a significant resource for axonal and DA neuronal cell biology, while the methodology developed here will enable future studies of other neural cell types. This mass spectrometry proteomics dataset is a part of "Subcellular proteomics of dopamine neurons in the mouse brain" (Hobson et. al, 2022)
Cell line construction and maintenance for Lyso-IP with or without genes linked with lysosomal storage disease
By onAssociated with the following preprint (published September 30th 2021): Progranulin deficiency results in reduced bis(monoacylglycero)phosphate (BMP) levels and gangliosidosis Sebastian Boland, Sharan Swarup, Yohannes A. Ambaw, Ruth C. Richards, Alexander W. Fischer, Shubham Singh, Geetika Aggarwal, Salvatore Spina, Alissa L. Nana, Lea T. Grinberg, William W. Seeley, Michal A. Surma, Christian Klose, Joao A. Paulo, Andrew D. Nguyen, J. Wade Harper, Tobias C. Walther, Robert V. Farese Jr. bioRxiv 2021.09.30.461806; doi: https://doi.org/10.1101/2021.09.30.461806
Immunohistochemistry Protocol – Chu lab (Wichman)
By onThis protocol describes immunohistochemistry.
Golgi immunopurification (Golgi-IP) for subcellular lipid profiling
By onThis protocol provides details for preparing Golgi-IP lipidomics samples.
pCAG-OSF-ATG13 (2-197)
By onPlasmid: To make ATG101/ATG13 HORMA complex for expression in mammalian cells.
FM1-43 endocytic uptake assay in HIPSC derived neurons
By onFM1-43 is a lipophilic styryl dye, nontoxic to the cells, virtually nonfluorescent in aqueous media. The dye will become fluorescent once it binds to the plasma membrane. In neurons that are actively releasing neurotransmitters, these dyes become internalized within the recycled synaptic vesicles, and the nerve terminals become brightly stained. This shows an increase in the fluorescence intensity of FM1-43. When the cells undergo another stimulation (through KCL or electrical) the FM1-43 dye containing vesicles will be released through exocytosis. Thus, resulting in a decrease in the FM1-43 fluorescence intensity.
Flag co-immunoprecipitation
By onProtocol used to pull down V5-FLAG-Gcase interacting proteins in HEK cells
Freezing of hPSCs grown on MEFs
By onThis protocol describes the standard procedure of freezing human pluripotent stem cells (hPSCs), which were grown on inactivated mouse embryonic fibroblasts (MEFs).
Rotarod Test to assess motor coordination in a mouse parkinsonian model
By onThis protocol evaluates motor coordination in the rotarod assay in parkinsonian mice.
Cryo-ET of S. cerevisiae during starvation (tomograms)
By onCryo-ET of S. cerevisiae during starvation (tomograms).
powereQTL – An R package for calculating sample size and power of bulk tissue and single-cell eQTL analysis
By onR package for calculating sample size and power of bulk tissue and single-cell eQTL analysis