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. 2005 May 10;388(Pt 1):7–15. doi: 10.1042/BJ20042033

Figure 1. Experimental strategy for sequential salt extraction of chromatin-associated proteins.

Figure 1

(A) Representation of the experimental strategy employing sequential salt extraction of HeLa nuclei with increasing salt concentrations; see the text for details. (B) Establishment of the conditions for generating DNA DSBs. HeLa cells were untreated (lanes 1 and 3), exposed to 25 Gy of γ-radiation (lane 2) or treated with 50 μM of etoposide (lane 4). The cells were collected after 3 h for nuclei isolation, histone extraction and immunoblot analysis for γ-H2AX expression. H2A was used as an internal control. (C) Protein extracts were prepared from HeLa cells in which DNA DSBs were induced as described above (M, molecular-mass markers; C, control; γ, γ-radiation; E, etoposide), and subjected to SDS/PAGE (12.5% gel) followed by silver staining. (D) The validity of the experimental strategy used was examined by immunoblotting for Ku proteins. Increased levels of Ku70 and Ku80 (results not shown), known to bind to the ends of broken DNA, were extracted from DSB-generated nuclei than from control nuclei at 0.5- and 0.65-M salt concentrations (lanes 7–12).