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A Scalable System for Identifying Changing Patterns of DNA -Binding Proteins During Stem Cell Differentiation /

Abstract

Transcription factors determine cell fate during differentiation and are capable of reprogramming distinct cell types. They are regulated by multiple mechanisms, many of which control their stability and their ability to bind to DNA. Although a variety of approaches allow for the detection of the changing landscape of transcription factors during differentiation, the majority of these screening approaches, eg. microarray, RNA sequencing, quantitative PCR, western blot, do not address whether the detected transcription factors are stable or even capable of binding to DNA. Moreover, antibodies are not available or are insufficient at characterizing thousands of different transcription factors. New advances have been made in nucleotide detection and these methods include end -labeled fluorescent dyes. Using this technology, we proposed an alternative electrophoretic mobility shift assay (EMSA) protocol using the Odessey® imaging system to supplement the shortcomings of the previously mentioned techniques. By using EMSA oligonucleotides with end- labeled fluorescent tags that can be visualized at a wavelength of 700nm with the Odessey® imaging system, results can be observed within three hours. This protocol provides a potentially scalable platform to screen for novel transcription factor binding profiles including those that function in embryonic stem cells or other cell lineages

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