New way to isolate simian virus 40 nucleoprotein complexes from infected cells: use of a thiol-specific reagent.
A new method for the isolation of simian virus 40 nucleoprotein complexes from nuclei of lytically infected cells is described. The method is based on the addition of a thiol-specific reagent, 5'5'-dithiobis(2-nitrobenzoic acid), to lysis and extraction buffers. By inhibiting an uncoating activity during simian virus 40 extraction, 5'5'-dithiobis (2-nitrobenzoic acid) allows the use of efficient extraction buffers, such as one containing Triton X-100 and EDTA, for the isolation of native simian virus 40 minichromosomes and virion-type structures. Use of the method is illustrated by following encapsidation of simian virus 40 minichromosomes in a pulse-chase experiment. Since 5'5'-dithiobis (2-nitrobenzoic acid) is an inhibitor of many different enzymes, the 5',5'-dithiobis (2-nitrobenzoic acid) extraction technique may be useful for the isolation of not only papovaviruses but also other viruses and possibly cellular chromatin.
D000818:Animals; D002467:Cell Nucleus / Q000032:analysis*; D002499:Centrifugation, Density Gradient; D002522:Chlorocebus aethiops; D004279:DNA, Viral / Q000302:isolation & purification*; D003852:Deoxyribonucleoproteins / Q000302:isolation & purification*; D004228:Dithionitrobenzoic Acid; D008854:Microscopy, Electron; D009698:Nucleoproteins / Q000302:isolation & purification*; D013539:Simian virus 40 / Q000032:analysis*; D014771:Virion / Q000032:analysis
D004279:DNA, Viral; D003852:Deoxyribonucleoproteins; D009698:Nucleoproteins; D004228:Dithionitrobenzoic Acid