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. 2012 Nov 2;23(3):295–302. doi: 10.1093/glycob/cws152

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© The Author 2013. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com.

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Fig. 2. — Purification of LARGE2dTM. (A) Schematic representation of LARGE and the LARGEdTM construct as described previously (Inamori et al. 2012), and LARGE2 and the LARGE2dTM construct used in the enzymatic activity assay. The transmembrane (TM) sequence of LARGE2 was replaced with a FLAG (3x) tag sequence, and the C-terminus was modified with myc and Hisx6 tags. CC, coiled-coil domain. (B) Expression and purification of test proteins. (Left) Immunoblotting with anti-FLAG antibody. For each stable clone, 50 μL of the culture medium was analyzed. pCMV9, cell clone obtained by transfection of empty vector. (Right) Purification of recombinant LARGE2dTM protein expressed in the culture medium. Proteins were run over the Talon metal affinity resin and eluted, and the fractions were analyzed by Coomassie brilliant blue (CBB) staining.