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- The greatest challenge in studying membrane proteins is the search for a solubilising medium. Detergents are the preferred membrane mimetic and although they can support membrane proteins, their ability to preserve the native state is ambiguous. We are currently investigating the use of a novel membrane mimetic, nanodiscs. Nanodiscs are lipid bilayer fragments encompassed by a protein scaffold. Membrane proteins can then be reconstituted directly into the nanodisc allowing for biochemical and biophysical characterisation. This technology has many intrinsic advantages over traditional proteoliposome approaches and detergent solubilised membrane proteins.
- Ligation Independent Cloning
- Traditional restriction enzyme and T4 DNA ligase based cloning approaches have been the staple of molecular biology based laboratories for over 2 decades. Ligation independent cloning allows us to bypass the majority of the time consuming and laborious steps in cloning DNA fragments. We have engineered a range of different protein expression constructs that share a common ligation independent cloning site. This allows us to rapidly and efficiently clone our PCR products directly into our these various expression vectors to determine the optimal construct for membrane protein expression.
- Rapid Western Detection
- Western blotting is an essential technique in protein expression studies, but it is very time consuming. We have recently explored the combination of dry-blotting coupled with a one-hour western detection approach. This has reduced the time taken from 5 hours to only 90 minutes (from completion of running the gel to ECL detection). This detection time is only marginally more than traditional Coomassie staining of an SDS-PAGE gel.
- Cell Disruption
- Cell disruption has been a traditional bottleneck for isolating cellular components. Rather than using multiple passage cell disruption techniques, such as a French Press, we have recently switched to a single passage cell disruptor. This streamlines the disruption procedure and allows us to rapidly proceed to the centrifugation steps.