Optimalization of southwestern technique for detection of binding of Epstein-Barr virus nuclear antigen 1 to origin of replication.
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.
Lin
J C
JC
Molecular Biology Section, National Center of Infectious Disease, Centers for Disease Control, Atlanta, GA 30333.
D000956:Antigens, Viral / Q000378:metabolism*; D001665:Binding Sites; D015139:Blotting, Southern; D015153:Blotting, Western; D002021:Buffers; D002478:Cells, Cultured; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019309:Epstein-Barr Virus Nuclear Antigens; D004854:Herpesvirus 4, Human / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D015636:Magnesium Chloride / Q000494:pharmacology; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
D000956:Antigens, Viral; D002021:Buffers; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D014764:Viral Proteins; D015636:Magnesium Chloride