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SMrT templates
Supported Membrane Templates (SMrT) are membranes organized in a variety of shapes resting on a passivated and PEG-cushioned glass surface. The most easily distinguishable forms are a planar supported bilayer and membrane nanotubes. Lipids in an organic solvent are spotted on a PEGylated glass coverslip and allowed to dry. The coverslip is then placed inside a flow cell and hydrated with buffer flow, which transforms the dry lipid into a planar lipid bilayer and an array of membrane tubes. Membrane tubes in these templates represent intermediates in a typical budding reaction and can be used to test candidate proteins to catalyze fission. Read-outs on membrane fission are clear and unambiguous (see movie) and their amenability to fluorescence microscopy allows the fundamental analysis of membrane fission pathways as well as discovery of protein machines that remodel membranes.
Use of the supported membrane tube assay system for real-time analysis of membrane fission reactions
Dar, Kamerkar and Pucadyil (2017) Nature Protocols
SUPER templates
SUPported bilayers with Excess membrane Reservoir (SUPER) templates are silica beads with a single lipid bilayer wrapped around but organized in folds. They are prepared by forming supported bilayers by the rupture and fusion of anionic liposomes on the silica surface in presence of high salt (see scheme below). They can be used in a variety of sedimentation-based applications from monitoring lipid-protein interactions to pulldowns to assaying membrane budding and fission. The excess reservoir can be visualized by adding them to a glass coverslip - once they settle, binding to the glass surface causes the excess reservoir to spill out and form a bilayer patch that looks like a sunny side-up.
Real-time visualization of dynamin-catalyzed membrane fission and vesicle release
Pucadyil and Schmid (2008) Cell
Pucadyil and Schmid (2010) Biophysical Journal
Analyzing membrane remodeling and fission using supported bilayers with excess membrane reservoir
Neumann et al. (2013) Nature Protocols
Sequential affinity chromatography (SAC)
Biochemical reconstitution requires rapid purification of large numbers of proteins to homogeneity. We achieve this by sequential affinity chromatography (SAC).
The gene for the protein of interest is engineered with a 6xHis tag at one end and a StrepII tag at the other end. Proteins are expressed in a system of choice and then purified according to the following scheme using an FPLC or a peristaltic pump. This consistently gives us highly pure and full length proteins in as little as ~ 1 hr.
SDS-PAGE showing results of
protein purification using SAC.
PLiMAP: proximity labeling-based identification of membrane-associated proteins
Biochemical reconstitution of membrane-associated phenomena requires analyzing the intrinsic lipid binding properties of proteins. We manage this by PLiMAP, which uses a patented novel bifunctional lipid (Patent 16/891373) incorporated in model membranes.
A facile, sensitive and quantitative membrane-binding assay for proteins
Jose, Gopan, Bhattacharyya, Pucadyil (2019) Traffic
PLiMAP: Proximity-Based Labeling of Membrane-Associated Proteins
Jose and Pucadyil (2020) Current Protocols in Protein Science
Pull-down matrices for analyzing protein-protein interactions on membrane surfaces
Discovery-based proteomics workflows that identify novel interactors rely on immunoprecipitations or pull-downs with genetically-tagged bait proteins immobilized on appropriate matrices. But strategies to analyse protein interactions on a diffusible membrane surface combined with the practical ease of pull-downs remain unavailable. Such strategies are important to analyse protein complexes that mature in composition and stability because of diffusion-based encounter between participant proteins. Here, we describe a generic pull-down strategy to analyse such complexes using chelating lipid-containing supported bilayers formed on silica beads. These templates can display desired His-tagged bait proteins on a diffusible membrane surface. Using clathrin-mediated endocytosis as a paradigm, we find that the clathrin-binding adaptor protein epsin1 displayed as bait on these templates pulls down significantly higher amounts of clathrin from brain lysates than when immobilized on conventional matrices. Together, our results establish the potential of such templates as superior matrices for analysing protein-protein interactions and resultant complexes formed on membrane surfaces.
Protein-protein interactions on membrane surfaces analysed using pull-downs with supported bilayers on silica beads
Andhare, Khurana, Pucadyil (2022)