We have optimized the conditions to purify protein complexes using antibodies against FLAG-tags 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 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 components of the 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. We have also been testing to the possibilities of obtaining high-resolution protein complex data by combining superpose-size fraction and IP-Mass Spectrometry.
| 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 |
