Isolation and identification of proteins binding to the major breakpoint region(mbr) of bcl2 gene

Abstract

Objective

We have previously found that mbr is a regulatory element of the bcl2 gene. The objective of this study is to isolate and identify the proteins binding to the 37 mbr in the 3′ -end of the mbr.

Methods

Streptavidin magnetic particles were ligated to concatameric oligonucleotides of 37 mbr and incubated with the nuclear extracts of Jurkat cells. The DNA-binding proteins were eluted and then resolved by SDS-PAGE. After silver staining, the protein bands were excised and subjected to MALDI-TOF MS.

Results

Several protein bands were detected after the isolation with magnetic particles, and Splicing factor, proline- and glutamine-rich(SFPQ), Poly(ADP-ribose) polymerase 1(PARP), and promyelocytic leukemia protein(PML) were identified by MALDI-TOF MS.

Conclusion

Several proteins were isolated and identified from the 37 mbr-protein complex. Results of this study establish a foundation for further study of the mechanisms by which mbr executes its regulatory function.