The following protocols are being standardized based on comparative studies of over 50 membrane proteins which have been purified intact from Escherichia coli, Pichia pastoris, insect, mammalian cells, and brain, fly and plant tissue using the SMALP system:
- Production of activated styrene maleic acid
- Optimization for pH for SMALPing membranes
- DIBMA protocol, Sandro Keller's talk, Cube Biotech DIBMA protocol, services and review
- Purification of SMALP-encapsulated proteins
- Solubilization of raft vs nonraft lipids from T cells
- SMA-PAGE and native gel electrophoresis
- Biophysical analysis of SMALPs by methods including:
differential scanning calorimetry
dynamic light scattering
fourier transform infrared
microfluidic diffusional sizing
13C NMR of phospholipase activity of PagP in SMALPs
1H,31P HSQC NMR of phospholipids in SMALPs
small angle neutron scattering
small angle X-ray scattering
- Flow cytometry, antigens and antibody studies with SMALPs
- Proteomic and lipid analysis of the SMALP'd metabolon
- Mass spectometry analysis of lipids and hENT1 and GlpG in SMALPs
- Structural analysis of SMALP-protein complexes by X-ray crystallography and electron microscopy and hi res structures of native AcrB and ACIII membrane assemblies.
Protocols are described in papers including Dafforn TR, Lee SC, Knowles TJ, Postis V, Jamshad M, Parslow R, Lin YP, Goldman A, Sridhar P, Overduin M, Muench SP. A Method for Detergent-free isolation of Membrane Protein with its Local Lipid Environment, Nature Protocols, 11, 1149-1162.
The discovery of SMALPs was first reported in 2009. Reviews on the progress since include:
- Overduin M, Esmaili M. Structures and Interactions of Transmembrane Targets in Native Nanodiscs. SLAS Discovery.
- Sun C, Gennis RB. Single-particle cryo-EM studies of transmembrane proteins in SMA copolymer nanodiscs. Chem Phys Lipids. 221:114-119
- Overduin M, Esmaili M. Native Nanodiscs and the Convergence of Lipidomics, Metabolomics, Interactomics and Proteomics. Appl. Sci., 9(6), 1230.
- Ravula T, Hardin NZ, Ramamoorthy A. Polymer nanodiscs: advantages and limitations. Chem Phys Lipids 219:45-49.
- Overduin M & Esmaili M. Memtein: the fundamental unit of membrane:protein structure and function Chem Physics Lipids. 218:73-84.
Join us for the next meeting which includes talks from a diverse array of experts and a practical session on SMALP protocols and R&D priorities for the next year.
Check out this video on the SMALP method by CUBE Biotech: