Xenbase Image ID: 123390

Figure 1. Purification and identification of the major 5′→3′ ssDNA exonuclease in Xenopus extracts. (A) Purification scheme. (B) Nuclease assay of the heparin flow through and 250 mM NaCl elute. The oligo substrate carried two 32P-labeled dA near the 3′-end and was attached to magnetic beads through a biotin at the 3′-end. After incubation at room temperature for 1 h, the reactions were terminated with 1% SDS, heated at 95°C for 5 min and then analyzed on an 8% TAE/PAGE. The 32P signal was detected by Phophoimager (Fuji). The nuclease activity was determined by calculating the fraction of small product generated (normalized to that of reaction 3 that contained both the 250 mM elute and the flow through). (C) A silver stained SDS–PAGE gel (4–12%) of the purified xDNA2 after the oligo beads purification. Load, proteins before binding to the oligo beads; unbound, proteins after incubation with beads; elute, bound proteins eluted by high salt buffer. (D) Nuclease assay of the purified xDNA2 in the presence of heparin flow through or buffer. The assay condition and the determination of the nuclease activity were similar to those in (B). Image published in: Liao S et al. (2008) © 2008 The Author(s). This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license Larger Image Printer Friendly View

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