Membrane preparation
We have established a number of classical protocols and methods for isolation of rather pure and intact membrane fractions from different types of tissue and cell cultures. Enrichment of specific compartments or organelles can be achieved by high-resolution density gradient centrifugation, free-flow electrophoresis (FFE) or partial solubilisation. Depending on the expression density of the target protein(s) and the purpose of the study, 20 mg to 2 g of tissue or 10
6-10
8 cells may be sufficient.
Solubilisation
Solubilisation from biological membranes is a necessary but critical step for analysis of membrane proteins. Since each protein and membrane environment has its specific biochemical properties, conditions have to be newly optimized for each case. We have established and routinely apply different assays to test for effective but gentle solubilisation:
- Western-based solubilisation screen to optimize efficiency
- Gel screen to investigate protein complex stability and size distribution
- Pulldown-assays to check for preservation of known protein-protein interactions
Using these assays in various projects (membrane proteomics and membrane protein-protein interactions) we have established more than 100 standardized buffers and conditions. These
ComplexioLyte buffers can be used for affinity purification of membrane protein complexes, native and denaturing isoelectric focusing, native gel electrophoresis and fractionation of membrane proteins for proteomic analysis. An overview of available buffers and applications is given in the
products section.
Affinity purification
We have developed proprietary methods that allow antibody-based affinity purification of protein complexes from solubilised native membranes. These methods work matrix-free, e.g. purification is carried out in solution, is in equilibrium (allowing capture of low-affinity binding partners) and has a low non-specific background. We have developed this further as PMAP (parallel micro affinity purification) technology that allows parallel purification of many different proteins or protein complexes from less than 2 ml of solubilisate in one experiment.
