Journal: FEBS Journal

Article Title: A new phospholipase A2 isolated from the sea anemone Urticina crassicornis - its primary structure and phylogenetic classification

doi: 10.1111/j.1742-4658.2010.07674.x

Figure Lengend Snippet: Fig. 1. Isolation of native UcPLA2. Native UcPLA2 was isolated from the exudate in a three-step chromatography procedure. (A) Gel filtration on a Superdex 75 HR 10 ⁄ 30 FPLC column in 10 mM ammonium acetate (pH 5.3) running buffer at a flow rate of 0.5 mLÆmin)1. Separation was repeated several times with 0.5 mL aliquots. (B) Hemolytic fractions were pooled and separated on a Mono S HR 5 ⁄ 5 cation exchange column at a flow rate of 1 mLÆmin)1 in the same ammonium acetate buffer as used for gel filtration. Bound proteins were eluted with a lin- ear gradient of 0.0–1.0 M NaCl. (C) Peak 3 fractions were subjected to RP-HPLC, using a Vydac 218TP C18, 4.6 · 250 mm, 7 lm reverse phase column. Proteins were eluted with an acetonitrile gradient of 0–70% (v ⁄ v) in 0.1% trifluoroacetic acid (v ⁄ v) at 1 mLÆmin)1. (D) A sam- ple of the cation exchange chromatography peak 3 separated on a 12.5% SDS ⁄ PAGE gel.

Article Snippet: Key fractions obtained by different types of chromatography were analyzed and checked for purity with the MiniProtean II SDS ⁄PAGE apparatus (BioRad, Hercules, CA, USA).

Techniques: Isolation, Chromatography