SMALP Protocols

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:
  circular dichroism
  differential scanning calorimetry
  dynamic light scattering
  fourier transform infrared
  microfluidic diffusional sizing and a video of the Fluidity One-W system
  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, Trieber C, Prosser RS, Picard LP, Sheff JG. Structures and Dynamics of Native-State Transmembrane Protein Targets and Bound Lipids. Membranes 11(6):451.
• Sun C, Gennis RB. Single-particle cryo-EM studies of transmembrane proteins in SMA copolymer nanodiscs. Chem Phys Lipids. 221:114-119
• Brown CJ, Trieber C, Overduin M, Structural biology of endogenous membrane protein assemblies in native nanodiscs, Curr Opin Struct Biol 69, 70-77
• Ravula T, Hardin NZ, Ramamoorthy A. Polymer nanodiscs: advantages and limitations. Chem Phys Lipids 219:45-49.

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:

Here are talks about SMALPs and membrane proteins by Alice Rothnie (Aston U) and Steve Harborne (Peak Proteins) and hosted by Nature Research.

Feel free to discuss below or post questions on Linkedin in or tweet @smalpnet.