Analysis of carbohydrate binding and phosphatase activity of Gg-laforin. A. His-tagged proteins were incubated with amylose resin, then the amylose resin was pelleted by centrifugation, the supernatant was removed, and SDS-PAGE buffer was added to the pellet to release proteins bound to the amylose resin. Proteins in the supernatant were precipitated, and SDS-PAGE buffer was added to the supernatant sample. Protein input (I), supernatant (S) and pellet (P) samples were resolved by SDS-PAGE and visualized by Western analysis. B. Specific activities of Gg-laforin and Hs-laforin were quantified against the artificial substrate pNPP at pH units 5.0-8.0. C. Phosphate release from amylopectin using Gg-laforin and Hs-laforin at pH units 5.0-8.0 was measured using a malachite green assay. Error bars indicate mean ± SD.
Brewer et al. BMC Biochemistry 2014 15:8 doi:10.1186/1471-2091-15-8