Immunocytochemistry – subcellular localization of ATP13A4
By onThis protocol describes an optimized method for visualizing the subcellular localization of over-expressed ATP13A4 in HeLa cells using immunocytochemistry.
IHC staining (DAB) of Iba1 and Gfap
By onThis protocol details the IHC staining (DAB) of Iba1 and Gfap.
Protein quantification by SDS-PAGE and Coomassie staining
By onThis protocol provides a step-by-step guide for quantifying protein concentration by SDS-PAGE and Coomassie staining.
Immunoblotting protocol
By onThis protocol outlines a step-by-step method for performing western blot analysis, including protein separation by gel electrophoresis, transfer to membranes, antibody incubation, and detection.
Expression and purification of ATP13A4 from Saccharomyces cerevisiae
By onThis protocol describes the expression and purification of human ATP13A4 with a C-terminal Twin-Strep tag from Saccharomyces cerevisiae using a CuSO₄-inducible system.
Measurement of ATP13A4 ATPase activity using ADP-Glo Max Assay
By onThis protocol describes a luminescence-based ADP-Glo Max assay (Promega) to measure the ATPase activity of ATP13A4 by detecting the rate of ATP hydrolysis.
Purification of ATP13A4 from HEK293T cells
By onThis protocol describes the purification of ATP13A4 from HEK293T cells stably overexpressing ATP13A4. The procedure includes cell lysis, membrane solubilization, and affinity purification using Strep-TactinXT resin.
Fluorescently labeled polyamine uptake (flow cytometry)
By onAssess polyamine uptake capacities of a specific cell line after incubation with fluorescently labeled polyamines and mean fluorescence intensitiy acquisition via flow cytometry.
Auto-(de)phosphorylation assay
By onProtocol measures ATP13A4 phospho-enzyme levels by detecting its phosphorylated intermediate on an aspartate residue. Utilizes SDS-PAGE under acidic conditions and autoradiography to detect -labeled intermediates.
RT-qPCR (cell lines)
By onThis protocol describes the use of RT-qPCR to assess msAtp13a4 mRNA expression following lentiviral knockdown in C8-D1A cells.
Fluorescently labeled polyamine uptake (confocal microscopy)
By onMethod visualizes polyamine uptake in cells using fluorescently labeled polyamines and confocal microscopy.
Collection of mouse cerebrospinal fluid from the cisterna magna
By onThis protocol describes the collection of mouse cerebrospinal fluid from the cisterna magna.
Preparation of total cell lysates
By onThis protocol outlines a step-by-step method for preparing cell lysates.
RNA isolation and cDNA synthesis
By onThis protocol details the isolation of RNA, purification, and subsequent cDNA synthesis for downstream applications such as qPCR.
RT-qPCR (primary cells)
By onProtocol details qPCR method for Atp13a4 expression in astrocytes and neurons using Fast SYBR Green Master Mix in a 96-well format. Normalization to 18S gene ensures accurate quantification for reliable gene expression analysis.
Developmental milestones assessment in newborn mice (P1–P21)
By onBehavioral tests for newborn mice from P1 to P21 assess weight gain, reflexes, and movements with a focus on developmental milestones.
Lentiviral vector production and primary astrocyte transduction
By onProtocol for lentivirus production & transduction into astrocytes is outlined, allowing efficient transduction & stable selection. Facilitates research on primary gene function in astrocytes.
Astrocyte morphology assessment in astrocyte-neuron co-cultures
By onThis protocol outlines the process for analyzing astrocyte morphology in astrocyte-neuron co-cultures using immunostaining and Sholl analysis.
Genotyping Atp13a4(KO) mice
By onProtocol for DNA extraction from mouse biopsies & genotyping Atp13a4(KO) mice involves alkaline lysis method, PCR with specific primers, & agarose gel analysis to differentiate genotypes.
