An efficient method for simultaneous isolation of biologically active transcription factors and DNA.
Transcription factors play a crucial role in gene regulation during different stages of eukaryotic development as well as in controlling various cellular disorders involving the immune system. In order to study the role of cellular DNAs and the effects of certain biologically active regulatory proteins, which can affect gene expression, we have developed a rapid and efficient method for preparing highly purified DNAs as well as nuclear and cytoplasmic proteins, simultaneously. These DNAs and proteins can be effectively analyzed to determine their genetic integrity and binding motifs to specific DNA sequences, respectively. This protocol avoids the drastic use of mechanical shearing of cells, aggressive use of detergents or high speed ultracentrifugation steps, as well as facilitating the ease of collecting samples in a sequential and effective manner with minimal time lapse during processing. 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 wash off the detergent and a high-salt buffer to extract nuclear proteins including transcription factors. The remainder of the cellular products are processed for DNA extraction. This method will be particularly useful to evaluate 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 or neoplastic disorders, with minimal physical contact to the laboratory personnel. This rapid DNA and protein isolation method can be widely used in various systems to analyze the modulation of DNA characteristics and transcriptionally active proteins as biomarkers in different human diseases.
Caruccio
L
L
Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595, USA.
Banerjee
R
R
D001483:Base Sequence; D002021:Buffers; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D015342:DNA Probes / Q000235:genetics; D003902:Detergents; D006801:Humans; D008722:Methods; D016328:NF-kappa B / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020836:Protein Structure, Quaternary; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D002021:Buffers; D015342:DNA Probes; D003902:Detergents; D016328:NF-kappa B; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D004247:DNA; D013755:Tetradecanoylphorbol Acetate