Verification of nucleolin copurification with HDGF. (A) Analysis of most abundant HDGFStrep-tag copurifying proteins by Coomassie stained SDS-PAGE. Proteins purified via Strep-Tactin®-MacroPrep matrices after transfection of cells with HDGFStrep-tag or untagged HDGF (Mock) were separated using SDS-PAGE and visualized by Coomassie staining. Visible proteins specific for HDGFStrep-tag sample correspond to Ku86 (86 kDa), nucleolin (110 kDa), PARP-1 (113 kDa) and the catalytic subunit of DNA-PK (460 kDa). (B) To confirm mass spectrometric results, lysates of HEK293 cells expressing HDGFStrep-tag fusion proteins and their eluate fractions from HDGFStrep-tag purification were examined by Western blot with a specific nucleolin antibody. Nucleolin is only present in eluates from cells expressing HDGFStrep-tag fusion proteins. (C) To investigate whether endogenous HDGF also interacts with nucleolin a cellular extract of untransfected HeLa cells was loaded onto a human HDGF antibody column. Non-specific proteins were washed out with 300 mM NaCl. Specifically binding proteins were eluted with 50 mM citric acid pH 3. Western blot analysis with a monoclonal anti nucleolin antibody confirmed copurification of nucleolin with endogenous HDGF.
Bremer et al. BMC Biochemistry 2013 14:2 doi:10.1186/1471-2091-14-2