Protocol Archive

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.

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.

This protocol describes behavioral testing to assess motor deficits in mice. It includes open field locomotor testing and scoring of levodopa-induced dyskinesia.

A method for assaying starvation-induced autophagy in HeLa cells that have been transfected with Halo-tagged constructs. This protocol was specifically used to investigate the impact of various mutations in the protein WIPI2 on starvation-induced autophagy in cells.

The original qualitative method was published in 2004 and measured the oligosaccharides selectively released from glycosphingolipids using a ceramide glycanase enzyme derived from the medicinal leech. This is an updated protocol with the focus on achieving sensitive and reproducible quantitation of glycosphingolipids from human plasma samples

An assay for measuring ULK1 kinase activity.

This protocol details about activation induced marker staining.

This protocol describes the procedure for detection of ATP10B in rat and mouse brain tissue by Western blotting

Step-by-step protocol for performing HyDrop-RNA. The duration of each step assumes an experienced protocol user. For a first-time user, we recommend doubling the expected time for each step.

Step-by-step protocol for execution of HyDrop-ATAC.

Protocol for producing microfluidic chips used in HyDrop experiment.

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.

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.

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.

This protocol describes the procedure for hippocampal neuronal cultures from new - born mouse pups.

An assay to study protein-protein interaction

This is a method for measuring protein enrichment on mitochondria in various conditions. In the resulting Western blot, one can assess the level of contamination of other organelles in the enrichment prep.

Protocol for producing dissolvable barcoded hydrogel beads used in HyDrop experiments.

This protocol transiently depletes TBK1 from HeLa cells and re-introduces a tagged TBK1 along with other relevant components of mitophagy. Because TBK1 is integral in many cellular processes, cells depleted of TBK1 exhibit poor health. Our protocol accomplishes ~70% depletion of endogenous TBK1 within 24 hours without excessive cell death.