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 spectrometry. 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 have recently carried out the mass spectrometry analyses of 24 TFs and are currently analyzing the data.
| 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 |
