Formaldehyde cross-linking of proteins to osteoclast differentiation factor promoter for the identification of biofunctional proteins.

OBJECTIVE To find the optimal cross-linking condition for nuclear factor (NF)Y (A, B, C), and find the transcription factors involved in osteoclast differentiation factor (ODF) expression. METHODS This experiment was carried out from 2002 to 2003 in Handa Lab, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Tokyo, Japan. To search for the NFY-binding condition, optimal formaldehyde cross-linking was detected, and the complex was purified by immunoprecipitation. Some proteins were detected as interactors. Interaction between NFYs and the ODF promoter was detected by polymerase chain reaction. RESULTS The optimal cross-linking condition for NFYs was determined using 10 samples with 0.7-1.3% formaldehyde, and 3-15 minutes incubation time. The results showed the interaction of NFY A, B, and C with each other for ODF promoter binding and involvement of other factors like vitamin D receptor in ODF expression. CONCLUSION Optimal cross-linking conditions vary, based on protein concentration, pH, additives, temperature, number of reactive groups, spacer arm length, and buffer volume and composition.