![]() ![]() However, the protocol for DNA FISH is often incompatible with protein immunohistochemistry it involves a DNA denaturation step that can reverse chemical crosslinks and denature protein epitopes, thus hindering primary antibody binding. To confirm whether a protein of interest is targeting a specific genomic locus, investigators have historically combined DNA fluorescent in situ hybridization (DNA FISH) with protein immunofluorescence (IF). As an alternative to ChIP-seq, investigators utilize microscopy to reveal protein localization, monitor biochemical interactions between proteins and DNA, and quantify binding mechanisms that lead to the formation of protein-DNA complexes. However, ChIP-seq involves several caveats: it is expensive, it requires access to sequencing platforms, and it is difficult to perform by inexperienced users. A common technique used to investigate protein-DNA localization is ChIP-seq, which captures positional information of proteins across the genome. The relationships between a DNA locus and the proteins that target that locus affect fundamental processes such as DNA replication, transcription, and repair. Overall, this study provides an alternative, accessible method for investigating protein-DNA interactions at the single gene level in Drosophila melanogaster polytene chromosomes. We demonstrate that this assay is sensitive enough to determine if our protein of interest, Multi sex combs (Mxc), localizes to single-copy target transgenes carrying histone genes. ![]() We developed a hybrid RNA FISH-IF protocol for use on Drosophila melanogaster polytene chromosome spreads in order to visualize colocalization of proteins and DNA loci. Our goal was to develop an alternative technique to investigate protein-DNA interactions by combining RNA FISH with IF. Additionally, combining DNA FISH with IF may be challenging for less experienced trainees. However, these assays are sometimes incompatible due to the required denaturation step in DNA FISH that can alter protein epitopes, hindering primary antibody binding. Combining DNA fluorescence in situ hybridization (FISH) with immunofluorescence (IF) is a quicker and inexpensive approach which has historically been used to investigate protein-DNA interactions in individual nuclei. Sequencing techniques such as ChIP-seq can yield genome-wide DNA binding profiles of transcription factors however this assay can be expensive, time-consuming, may not be informative for repetitive regions of the genome, and depend heavily upon antibody suitability. Investigating protein-DNA interactions is imperative to understanding fundamental concepts such as cell growth, differentiation, and cell development in many systems. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |