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Neuroscience

Categories
+ Basic technology
+ Behavioral neuroscience
- Cellular mechanisms
Cell adhesion
Cell isolation and culture
Extracellular matrix
Intracellular signalling
Lymphatic vessel
Microglia
Mitochondria
Myelin
Neuronal fate
Protein isolation
Receptor-ligand binding
RNA Localization
Synaptic physiology
Tissue isolation and culture
+ Development
+ Nervous system disorders
+ Neuroanatomy and circuitry
+ Peripheral nervous system
+ Sensory and motor systems
Protocols in Past Issues

Using Protein Painting Mass Spectrometry to Define Ligand Receptor Interaction Sites for Acetylcholine Binding Protein
AG Alexandru Graur
NE Natalie Erickson
NK Nadine Kabbani
Q&A 0 Q&A
1992 Views
Jan 20, 2025

Nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channels expressed in nervous and non-nervous system tissue important for memory, movement, and sensory processes. The pharmacological targeting of nAChRs, using small molecules or peptides, is a promising approach for the development of compounds for the treatment of various human diseases including inflammatory and neurogenerative disorders such as Alzheimer’s disease. Using the Aplysia californica acetylcholine binding protein (Ac-AChBP) as an established structural surrogate for human homopentameric α7 nAChRs, we describe an innovative protein painting mass spectrometry (MS) method that can be used to identify interaction sites for various ligands at the extracellular nAChR site. We describe how the use of small molecule dyes can be optimized to uncover contact sites for ligand–protein interactions based on MS detection. Protein painting MS has been recently shown to be an effective tool for the identification of residues within Ac-AChBP involved in the binding of know ligands such as α-bungarotoxin. This strategy can be used with computational structural modeling to identify binding regions involved in drug targeting at the nAChR.

Assessing Gαq/15-signaling with IP-One: Single Plate Transfection and Assay Protocol for Cell-Based High-Throughput Assay
Élie Besserer-Offroy Élie Besserer-Offroy
RB Rebecca L Brouillette
JL Jean-Michel Longpré
PS Philippe Sarret
Q&A 0 Q&A
5594 Views
Aug 20, 2020
Cell-based functional assays are an important part of compound screening and drug lead optimization, and they can also play a crucial role in the determination of the residues involved in ligand binding and signaling for a particular G-protein-coupled receptor. Conventional methods used for Gαq/15-coupled receptors rely on the use of fluorescent probes for Ca++ sensing (such as Fura-2 and Fluo-4) or on the incorporation of [3H]-inositol into inositol 1,4,5- triphosphate (IP3). However, these methods are not suitable for screening large libraries of compounds or for screening several mutants of the same receptor. In contrast, the IP-One assay by Cisbio is a TR-FRET assay suitable for large compound library screening when using stable cell lines that express a specific 7TMR. However, when using transiently transfected mutants of a 7TMR, this assay is not ideal, as it requires a two-step protocol of cell culture. Therefore, we have optimized the IP-One assay protocol using the reverse transfection method in 384-well plates. This offers a time- and resource-efficient alternative to the two-step protocol previously used for the screening of several mutants of Gαq/15-coupled 7TMRs.

Supported Cell Membrane Sheets to Monitor Protein Assembly
SE Simon Erlendsson
TT Thor Seneca Thorsen
KM Kenneth Lindegaard Madsen
Q&A 0 Q&A
5655 Views
Sep 20, 2019
Studying protein-protein and protein-lipid interactions in their native environment is highly desirable, yet, the heterogeneity and complexity of cellular systems limits the repertoire of experimental methods available. In cells, interactions are often taking place in confined microenvironments where factors such as avidity, hindered diffusion, reduced dimensionality, crowding etc. strongly influence the binding kinetics and therefore it can be problematic to equate binding affinities obtained by bulk in-solution methods (e.g., Fluorescence Polarization, Isothermal titration calorimetry, Microscale thermophoresis) with those occurring in real cellular environments. The Supported Cell Membrane Sheet method presented here, addresses these issues by allowing access to the inner leaflet of the apical plasma membrane. The method is a highly versatile, near-native platform for both qualitative and quantitative studies of protein-protein and protein-lipid interactions occurring directly in or on the plasma membrane.

Visible Immunoprecipitation (VIP) Assay: a Simple and Versatile Method for Visual Detection of Protein-protein Interactions
Yohei Katoh Yohei Katoh
KN Kentaro Nakamura
KN Kazuhisa Nakayama
Q&A 0 Q&A
13714 Views
Jan 5, 2018
The visible immunoprecipitation (VIP) assay is a convenient alternative to conventional co-immunoprecipitation (Katoh et al., 2015). By processing lysates from cells co-expressing GFP-fusion and RFP-fusion proteins for immunoprecipitation with GST-tagged anti-GFP Nanobody and glutathione-Sepharose beads, protein-protein interactions can be visualized by directly observing the beads bearing immunoprecipitates under a fluorescence microscope. This assay can examine a large number of protein combinations at one time, without requiring time-consuming procedures, including SDS-PAGE and immunoblotting. Furthermore, the VIP assay can examine complicated one-to-many and many-to-many protein interactions. Another important point of the VIP assay is the use of nanobodies for immunoprecipitation. A Nanobody is a single-domain antibody derived from Camelidae (camels and relatives). Because of its small size, high-affinity, high-specificity, and stability, anti-GFP Nanobody expressed in E. coli can be purified on a large scale, and used virtually inexhaustibly for immunoprecipitation experiments. Here we describe protocols for preparation of GST-tagged anti-GFP Nanobody and the VIP assay.

Adenosine A2A Receptor Ligand Binding Experiments by Using Real-time Single-cell FRET
VF Víctor Fernández-Dueñas
Kenneth A. Jacobson Kenneth A. Jacobson
FC Francisco Ciruela
Q&A 0 Q&A
11061 Views
Mar 20, 2014
We designed a fluorescence resonance energy transfer (FRET)-based approach to study the ligand binding constants of the adenosine A2A receptor (A2AR). Our assay is based in the interaction of a fluorescent A2AR agonist ligand (MRS5424) with an A2AR tagged with the cyan fluorescent protein (CFP) at the N-terminus (i.e. A2ARCFP) and expressed in living cells. Thus, upon fast superfusion of the A2ARCFP expressing cells with MRS5424, the ligand-receptor interaction is determined by single-cell FRET in a real-time mode. Accordingly, our approach allowed immediate ‘real-time’ readout of the ligand-receptor interaction, thus allowing kinetic binding experiments, a feature impossible to achieve using conventional radioisotope-labelled ligands. In addition, since our assay permitted the visual confirmation of receptor localization it also allowed localized saturation binding experiments.

Autoradiographic 3H-Gaboxadol Receptor Binding Protocol
LL Lynne Ling
Donald Caspary Donald Caspary
Q&A 0 Q&A
7942 Views
Dec 5, 2013
Gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, THIP), a GABAA receptor δ-subunit specific agonist, when present at low (μM) concentrations, preferentially binds and activates extrasynaptic (non-γ2, δ-subunit-containing) GABAARs (Storustovu and Ebert, 2006; Richardson et al., 2011, 2013).

In this prototype saturation binding experiment, a series of concentrations of [3H]gaboxadol (5, 10, 25, 50, 75, 100, 250 and 400 nM) will be used. GABA at 200 μM will be added into binding mixtures as a cold displacer for [3H]gaboxadol. Slide mailers are used and each requires 7 ml binding mixture. Pre-, post-washing and binding buffer is 50 mM Tris-Citrate (pH 7.1). The detailed procedure is outlined below.