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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAAG000703-03
Application #
8148292
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2010
Total Cost
$481,439
Indirect Cost
Name
National Institute on Aging
Department
Type
DUNS #
City
State
Country
Zip Code