An efficient method for simultaneous isolation of biologically active transcription factors and DNA

Transcription factors play a crucial role in gene regulation during differe nt stages of eukaryotic development as well as in controlling various cellu lar disorders involving the immune system. In order to study the role of ce llular DNAs and the effects of certain biologically active regulatory prote ins, which can affect gene expression, we have developed a rapid and effici ent method for preparing highly purified DNAs as well as nuclear and cytopl asmic proteins, simultaneously. These DNAs and proteins can be effectively analyzed to determine their genetic integrity and binding motifs to specifi c DNA sequences, respectively. This protocol avoids the drastic use of mech anical shearing of cells, aggressive use of detergents or high speed ultrac entrifugation steps, as well as facilitating the ease of collecting samples in a sequential and effective manner with minimal time lapse during proces sing. Such an approach permits the analysis of a large number of samples in a short time. The current technique uses a non-ionic detergent to isolate nuclei, and obtain the cytosolic extract, a low-ionic strength buffer to wa sh off the detergent and a high-salt buffer to extract nuclear proteins inc luding transcription factors. The remainder of the cellular products are pr ocessed for DNA extraction. This method will be particularly useful to eval uate the time course effects of various cell signal transducing biological modifiers such as cytokines or mitogens, as well as drugs used in therapy, especially in infectious diseases and also in immunological qr neoplastic d isorders, with minimal physical contact to the laboratory personnel. This r apid DNA and protein isolation method can be widely used in various systems to analyze the modulation of DNA characteristics and transcriptionally act ive proteins as biomarkers in different human diseases. (C) 1999 Elsevier S cience B.V. All rights reserved.