Hilger et al. (2002)  purified small quantities of alpha- and beta-parvalbumins from Rana esculenta and Rana spp. by SDS-PAGE (18% acrylamide) in a MiniPrep Cell (Bio-Rad) for microsequencing. Recombinant alpha- and beta-parvalbumins were expressed in Escherichia coli with an N-terminal 6-histidine tail. The recombinant proteins were purified by Nickel-nitrilotriacetic acid (Ni-NTA) resin (Qiagen, Hilden, Germany) under denaturing conditions.
Hamada et al. (2004)  purified parvalbumins from bullfrog skeletal muscle, homogenized with 3 volumes of 0.15 M NaCl in 0.01 M phosphate buffer, pH 7.0, and centrifuged at 18000 x g for 20 min. at 4° C. The supernatant was concentrated in vacuo and chromatographed on a Sepadex G-75 size exclusion column equilbrated with 0.15 M NaCl in 0.01 M phosphate buffer, pH 7.0. Fractions containing parvalbumin were identified by ELISA, pooled and applied to reverse phase HPLC on a TSKgel ODS-120T column (0.46 x 25cm; Tosoh, Tokyo, Japan). Fractions were eluted at 1 ml/mi. with 0.1% trifluoracetic acid for 5 min., followed by a linear gradient of acetonitrile (0.0-38.5% in 2 min., 38.5-45.5% in 60 min. and 45.5-70% in 2 min.) in 0.1% (v/v) trifluoracetic acid. Alpha- and beta-parvalbumin were separated and identified by N-terminal and peptide sequencing (beta-parvalbumin has a blocked N-terminus).