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.