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  • Title: Optimalization of southwestern technique for detection of binding of Epstein-Barr virus nuclear antigen 1 to origin of replication.
    Author: Lin JC.
    Journal: Biochem Biophys Res Commun; 1993 Mar 15; 191(2):721-6. PubMed ID: 8384851.
    Abstract:
    An optimized protocol of Southwestern analysis for detection of Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) binding to the origin of viral replication (oriP) was described. The unique feature of this optimized protocol includes the restoration of the denatured proteins to native conformation after SDS-polyacrylamide gel electrophoresis and blocking of nonspecific and low-affinity binding sites prior to DNA binding. The parameters and conditions that may affect the specific interaction of EBNA-1 to oriP DNA were then determined. The specific binding was affected by divalent cations (MgCl2) and ionic strength; the optimal concentrations of MgCl2 and NaCl were observed at 10 and 300 mM, respectively. The various buffer systems and pH values tested had no apparent effect on EBNA-1 binding. Under the optimal conditions, a single protein of 68 Kd was detected and the bindings of other nonspecific, low-affinity DNA-binding proteins were abolished. The authenticity and specificity of 68 Kd protein as EBNA-1 were determined by reaction with antibody specific for EBNA-1 and competition assay with specific DNA sequence. After optimization this technique can be a powerful and yet simple means of studying protein-DNA interactions and their roles in gene expression.
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