Feb 01, 2023
We use this protocol and it’s working
Created: Feb 01, 2023
Last Modified: Feb 01, 2023
PROTOCOL integer ID: 76248
Keywords: ASAPCRN, LRRK2, Rab29, Golgi, immunofluorescence
- 1Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK
Abstract
Previous studies using confocal fluorescence microscopy showed that transient over-expression of Rab29 recruits the bulk of cellular LRRK2 to the Golgi, which results in its activation (PMID: 29212815). Here we describe our confocal immunofluorescence microscopy method for measuring the co-localization of LRRK2 and Rab29 in a cell-based assay. This method can be used to screen the impact that LRRK2 mutations or Rab29 mutations have on LRRK2 recruitment to the Golgi, as well as the effect of any compound on Rab29-induced LRRK2 recruitment to the Golgi.
Note: This protocol can be used for HEK293 cells and HeLa cells. For other cell types, experimental conditions may need optimising.
Guidelines
This protocol can be used for HEK293 cells and HeLa cells. For other cell types, experimental conditions may need optimising.
MATERIALS
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HEK293 cells (ATCC #CRL-1573) cultured in complete growth medium: DMEM (Thermofisher Scientific #11960-044) supplemented with 10% Fetal Calf Serum, qualified, Brazil (Thermofisher Scientific #10270-106), penicillin/streptomycin (Thermofisher Scientific #15140-122) and L-glutamine (Thermofisher Scientific #25030-024)
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N-terminus GFP-tagged LRRK2 (wild-type or mutant) cDNA in a pCMV5 vector; HA-empty vector and HA-tagged Rab29 (wild-type or mutant) cDNA in a pCMV5 vector. All plasmids used for our studies are available from the MRC PPU Reagents and Services (mrcppureagents.dundee.ac.uk).
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Polyethylenimine (PEI) “Max” (Linear, Mw 40,000) (Polysciences, Inc., #24765); 1 mg/ml (w/v) solution in milliQ water, pH 7.4; sterile filtered.
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Opti-MEM Reduced Serum Medium (ThermoFisher Scientific #31985062)
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Glass coverslips (22mm x 22mm squared glass coverslips)
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Tissue culture-treated flat bottom cell culture 6-well plates (Thermo Scientific Nunc #142475)
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Optional: Poly-L-Lysine solution, mol wt 150,000-300,000, sterile-filtered (Sigma, P4832)
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4% (w/v) paraformaldehyde in PBS, pH 7.4 (Alfa Aesar by Thermo Fisher Scientific, J61899). Note: This must be methanol-free.
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Phosphate buffered saline (PBS), pH 7.4 (ThermoFisher Scientific #10728775)
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NP-40 alternative (Merck #492016)
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Bovine Serum Albumin (BSA) (Sigma-Aldrich #A7906)
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Primary antibodies: Anti-HA tag mouse monoclonal antibody [HA.C5] (Abcam, ab18181) and anti-ACBD3 rabbit polyclonal antibody (Sigma-Aldrich, HPA015594). Optional: Anti-GFP chicken polyclonal antibody (Abcam, ab13970)
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Secondary antibodies: Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Alexa Fluor™ 568 (Invitrogen A11031) and Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Alexa Fluor™ Plus 647 (Invitrogen A32733). Optional: Goat anti-Chicken IgY (H+L) Secondary Antibody, Alexa Fluor™ 488 (Invitrogen A-11039)
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DAPI (bisBenzimide H 33342 trihydrochloride) (Sigma Aldrich #B2261)
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Vectashield Antifade mounting medium (Vector Laboratories, H-1000)
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Glass microscope slides
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CO2incubator for growing cells
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Laminar flow hood for cell culture
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Zeiss confocal laser scanning microscope
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