Protocol Archive

Protocol for labeling a purified protein with an NHS ester fluorescent dye.

Assess endocytic rate in cells using tagged transferrin (Alexa647) and flow cytometry readout.  

Assess polyamine uptake capacities of a specific cell line after incubation with fluorescently labeled polyamines and mean fluorescence intensity acquisition via flow cytometry. Watch an interview about this protocol with Mujahid Azfar, MSc.

To validate a genome wide CRISPR screen, the authors select the top hits and create lentiviruses to validate the hits. Rather than screening each virus from single cell clones, the authors analyze the infected cells as pools according to this protocol.  

This protocol describes a pooled, CRISPR Cas9 screen to identify modulators of LRRK2 activity. The CRISPR-Cas9 based screen is carried out in mouse cells using a ready-to-use pooled guide RNA (gRNA) mouse library consisting of 78,637 gRNAs targeting 19,674 genes and an extra 1,000 control gRNAs.

This bioinformatics approach has been taken to investigate the interactome of the NSL complex, to unpick its relevance to Parkinson’s disease progression. The mitochondrial interactome of the NSL complex has been built, mining 3 separate repositories: PINOT, HIPPIE, and MIST, for curated, literature-derived protein-protein interaction (PPI) data.  

This protocol provides details for analyzing GolgiIP lipidomics samples using liquid chromatography mass spectrometry (LC-MS) for nonpolar lipid profiling.

This protocol provides details for preparing Golgi-IP lipidomics samples.  

This protocol provides details for analyzing GolgiIP metabolomics samples using liquid chromatography mass spectrometry (LC-MS) for polar metabolite profiling.

This protocol provides details for preparing Golgi-IP metabolomics samples.  

MST kinase phosphorylates Rab proteins are at the same site as LRRK2 and have been used to phosphorylate Rab8A and Rab10 quantitatively. This protocol includes a method to produce phosphoRab8A protein and remove as much contaminating MST3 as possible, to enable use of the phosphoRab to test subsequent activation of LRRK2 kinase.  

The LRRK2 Armadillo domain contains multiple Rab GTPase binding sites. To show that the sites can be occupied simultaneously, we use this assay. The idea is to immobilize Rab8A, bind Armadillo domain, and test if phosphoRab10 can bind to Rab8A-immobilized Armadillo domain.

This protocol provides a technique to determine the radiolabeled polyamine uptake capacity in cells, via the acquisition of disintegrations per minute (DPM) using a Liquid Scintillation Counter.  

This protocol describes how Golgi, isolated from cells by GolgiTAG immunoprecipitation (IP) (as described in dx.doi.org/10.17504/protocols.io.6qpvrdjrogmk/v1), can be prepared and imaged using TEM. This protocol can also be used to image any organelles isolated using various organelle-IP protocols that are available.

This method that can be used to verify the correct localisation of HA-tagged Golgi proteins (like TMEM115-3HA, or GolgiTAG), by assessing their colocalisation with known Golgi markers such as GM130, GOLGIN97 and ACBD3 or can be used to assess if HA-tagged, non-Golgi annotated proteins transiently expressed in cells localise at the Golgi.

This protocol describes an immunoblotting method to assess efficient enrichment of Golgi proteins in Golgi immunoprecipitation products compared to whole cell lysates and the purity of the immunoprecipitated Golgi by monitoring the expression of other organelles’ markers. This method can also be used to verify the expression of GolgiTAG (TMEM115-3HA) in cells.

This protocol details how to generate stable cell lines using a retrovirus system (can also be used for lentivirus systems) and includes the production of retroviruses encoding the transgene of interest in HEK293FT and the subsequent transduction of the cell line that is intended to stably express the protein of interest.

This protocol describes the steps to use experimentally validated human data to create a protein-protein interaction network (PPIN) based on disease causative genes. Network analysis (combination of topological functional analyses) will lead to the identification of biological processes relevant for disease and disease endophenotypes.

This is a method for imaging cells and quantifying subcellular structures in the resulting data. We developed this protocol for assessing clearance of mitochondria involving OPTN and TBK1, however the method could be used for other applications.

Protocol for cylinder behavioral test for rats.