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Figure 3 | BMC Biochemistry

Figure 3

From: A ~35 kDa polypeptide from insect cells binds to yeast ACS like elements in the presence of ATP

Figure 3

Identification of p35. (A) Western blot analysis of salt eluate and reduced GSH eluate using anti GST antibody. GST-Cdc6 beads were washed using wash buffer containing NaCl (200 mM and 300 mM respectively). Beads following salt wash were treated with reduced glutathione and released proteins were run in SDS-PAGE along with salt eluate followed by western blot analysis. GST-Cdc6 was found only in GSH eluate. (B) Silver staining of salt eluate: Salt eluates were run in SDS-PAGE and proteins were visualised by silver stain. A distinct band of p35 was found both in 200 mM and 300 mM salt eluate. Molecular mass markers are shown on the right. (C) DNA binding activity of p35. Gel shift assays were performed using salt wash (300 mM NaCl eluate) and GSH eluate following 300 mM salt wash of GST-Cdc6 beads. ARS 240 bp DNA fragment was used as probe. 300 mM salt eluate gave a strong band shift whereas GSH eluate failed to do so though it contained GST-Cdc6 as shown in figure 3A.

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