DNA mobility shift assay coupled with SDS-PAGE for detection of DNA-binding proteins.

DNA mobility shift assay is widely performed to analyze sequence-specific DNA-binding proteins in cellular extracts (2), and the DNA-binding proteins can be identified by use of antibodies that induce further retardation of probes (supershift). Gel extracts from a shifted band contain not only DNA segments and DNA-binding proteins but also nonspecific proteins located at the same position in the gel, and this makes it difficult to identify a DNA-binding protein of interest in the gel extracts. In this study, a modified DNA mobility shift assay coupled with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was designed to overcome this difficulty. Epstein-Barr virus (EBV) nuclear antigen1 (EBNA-1), which binds specifically to the EBV DNA latent replication origin, oriP, and is essential for replication of EBV DNA (3,5) was used as a DNA-binding protein to demonstrate this new method. The probe used was oriP labeled with digoxigenin (DIG) and [α-32P]dCTP ([32P]DIG-oriP), and an antiDIG antibody was used as a supershift reagent. The mobility shift of [32P]DIG-oriP by the K2 polypeptide derived from EBNA-1 is demonstrated in Figure 1A (lane 2), together with the further retardation of [32P]DIG-oriP resulting from addition of anti-DIG antibody (supershift: lane 4). To analyze the components of the shifted and supershifted bands, each lane of the DNA mobility shift gel was cut out and soaked in SDS-PAGE sample buffer containing 2% SDS for 15 min to dissociate DNA-protein complexes in the gels. Each gel slice was placed horizontally onto a stacking gel of SDS-polyacrylamide (Figure 1, B and C), and SDS-PAGE was run according to Laemmli (4). Separated proteins were detected with the Silver Stain Kit (WAKO, Tokyo, Japan), and the migration patterns in SDS-polyacrylamide gels were compared. As shown in Figure 1B, K2 (closed triangle) was detected at a position consistent with its molecular mass (21 kDa) and with its BENCHMARKS