Quikchange

The full length Hsp20 sequence was cloned into a pET28c vector (Novagen) in order to express an N-terminal His-tag and then transformed into competent BL21 cells (Invitrogen, Paisley). Cells were grown until OD₆₀₀ ~ 1, 1 M of IPTG was then added and cells were grown for a further 24 h at 37 °C. The protein was then purified using nickel affinity chromatography. The resulting protein product was then checked for impurities on a 4–12% gel and then verified through western blotting techniques. Subsequent site-directed mutagenesis of this vector was carried out using Quikchange (Stratech) in accordance with manufacturer's protocol.

Full-length Hsp20 was cloned into pcDNA3.1/V5-His-TOPO vector (Invitrogen) and related mutants created using Quikchange (Stratech). The various Hsp20 constructs and an empty vector control were electroporated into SH-SY5Y cells using nucleofection kit V (Amaxa) in accordance with manufacturer's instructions. Cell were seeded at a density of 5 × 10³/well into seeded into 96-well plate for MTT-based assays or 96-well E-plate for xCELLigence based assays and left overnight to allow for cell re-attachment. Remaining cells were seeded into 6 well plates and harvested after 48 h to confirm expression of the various Hsp20 constructs. Addition of Aβ peptides and vehicle controls was carried out once cell index reached 1. The xCELLigence SP system (Acea) was used for real-time monitoring of cell growth for a minimum of 48 h post addition of Aβ peptides. The resulting data was analysed using (RTCA) real-time cell analyzer software (Roche) and exported to Excel. MTT based cell viability was carried out in parallel in accordance with Promega CellTiter 96® non-radioactive cell proliferation assay (G4000) in accordance with manufacturer's protocols and added 48 h post addition of Aβ peptides.