Purification and stability analysis of Hs-laforin and Gg-laforin. A. Hs-laforin was expressed in E. coli and purified by affinity chromatography in the absence (-) and presence of 15% maltose or 10 mM β-cyclodextrin (BCD). Fractions of the pellet (P) and supernatant (S) after high-speed centrifugation and fractions of the IMAC elution (E) were analyzed by SDS-PAGE and stained with Coomassie Blue dye. B. Elution fractions from Hs-laforin and Gg-laforin preparations were concentrated using centrifugal filter units. Volume and concentration of each preparation were monitored throughout centrifugation, and protein concentration was measured using a Bradford assay. Total protein content for each preparation before and after concentration and the percent reduced were calculated for each preparation. C. Elution fractions were concentrated to approximately 2-4 mg/ml and incubated at room temperature for eight days. The concentration of each protein was measured during the course of the experiment using a Bradford assay.
Brewer et al. BMC Biochemistry 2014 15:8 doi:10.1186/1471-2091-15-8