Microplate-nucleotide Hybridization Based Elisa-like Assay For Detection Of DNA Binding Protein

Transcription factors, also known as sequence-specific DNA binding proteins,contain one or more DNA binding domains (DBDs). They are located in cytoplasm. In physiological condition or induced by a specific stimulus, transcription factors would be transported into the nucleus from cytoplasm, and then bind to trans-acting factors such as promoters, enhancers or attenuators, thereby up-regulating or down-regulating transcription of adjacent gene. Therefore, the determination of DNA binding activities of transcription factors is pivotal to our understanding of cellular functions. Currently used methods are all based on the interactions between DNA binding proteins and their counterparts, that is to say, use the trans-acting factors as probe to detect the DNA-binding activities of transcription factors. Because transcription factors can prevent specific double-stranded nucleic acids from being eluted to single-stranded in high stringency condition, herein, we detect the protected double-stranded nucleic acids to determine the DNA-binding activities of transcription factors indirectly. Microplate-nucleotide hybridization based ELISA-like assay did not require complex instruments and complicated procedure, and could be applied to multiplex detection without the usage of specific antibody in short time. We have used the method to demonstrate the DNA-binding activity of c-Jun, a well-known model of inductive transcriptional regulatory responses. But the method is easily adaptable for the study of any transcription factor. This allows rapid and accurate detection of as low as 3.5ng of purified c-Jun protein or 0.625ug crude cell nucleus extract. Owing to the rapidness, ease, high sensitivity and selectivity of the method, the microplate-nucleotide hybridization based ELISA-like assay may be used in various applications and research fields where quantitative, cost-effective and large-scale measurements of the DNA-binding activities of transcription factors are required, including screening of responses in multiple treatments in cellular and molecular biology, evolutionary biology, environmental monitoring, and drug discovery.