We have optimized the conditions to purify protein complexes using antibody against FLAG-tag from mouse ES cells. We used mouse ES cells, where a FLAG-tagged transcription factor can be overexpressed in the absence of tetracycline for 72 hours. The purified protein complex was first analyzed by silver-stained gels and subsequently analyzed by mass spectrophotometry. The results indicate that our procedure works well. For example, we have used a FLAG-antibody to isolate a putative protein complex from the Cdx2-manipulated ES cells. Mass spectrometric analysis of the immunoprecipitated protein complex revealed a number of proteins matched by multiple peptides. Based on the relatively high number of peptide matches, the following protein groups are likely to be the components of CDX2 complex: (i) NuRD (nucleosomal remodeling and histone deacetylase) complex, including HDAC1, MBD3, and CHD4;(ii) SALL4, (iii) PARP1;and (iv) KPNB1 (Importin-1-beta) - a protein known for its function in nuclear transport. We are currently scaling up the procedure to analyze protein complexes for the initial 60 TFs. To aid in the protein complex analysis, we have also developed a database of mouse protein-protein interactions by transferring the protein-protein interaction information experimentally obtained from other model organisms.
| Sharov, Alexei A; Nishiyama, Akira; Piao, Yulan et al. (2011) Responsiveness of genes to manipulation of transcription factors in ES cells is associated with histone modifications and tissue specificity. BMC Genomics 12:102 |
| Nishiyama, Akira; Xin, Li; Sharov, Alexei A et al. (2009) Uncovering early response of gene regulatory networks in ESCs by systematic induction of transcription factors. Cell Stem Cell 5:420-33 |
