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Fig. 1 | BMC Molecular Biology

Fig. 1

From: Novel insight into the composition of human single-stranded DNA-binding protein 1 (hSSB1)-containing protein complexes

Fig. 1

Immunoprecipitation of hSSB1 from samples enriched for non-soluble nuclear proteins. a, b HeLa cells were lysed in a buffer containing 10 mM KCl and nuclei collected by centrifugation. Soluble-nuclear proteins were then extracted from these nuclei by incubation in a buffer containing 420 mM NaCl. The remaining precipitate (containing non-soluble nuclear proteins) was digested in a buffer containing the nuclease, benzonase. The soluble and non-soluble nuclear protein fractions (10 μg) were separated by PAGE and immunoblotted with antibodies against hSSB1, nucleolin (soluble-nuclear protein marker) and H3 (chromatin-bound non-soluble nuclear protein marker). c 500 μg of non-soluble nuclear protein was incubated overnight at 4 °C with protein G dynabeads bound to a hSSB1 antibody, or a sheep isotype control IgG. Beads were washed five times and protein eluted by heating to 80 °C in 3× SDS loading buffer for 5 min. 10% of the eluent was separated by PAGE and stained overnight in colloidal coomassie brilliant blue G-250. The red 28 kDa marker indicates the migration of hSSB1. d The remaining eluent was resolved on a 10% acrylamide SDS-PAGE gel to a depth of 8 mM and stained overnight in colloidal coomassie brilliant blue G-250

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