Type of Submission
Poster
Keywords
Tetrahymena, mitotic regulation, H3K27, acetylation, gene regulation
Proposal
Histone acetylation is critical for gene expression in eukaryotes. Acetylated lysines on histone tails are bound by bromodomain containing proteins which recruit chromatin remodeling proteins, site-specific transcription factors and other transcriptional apparatus. Our lab is particularly interested in the interactions between histone H3 acetylated at lysine 27 (H3K27ac) and any proteins that might be binding to it. In our previous work, we immunoprecipitated (using CO-IP) whole Tetrahymena extract with an anti-H3K27ac antibody and used an antibody binding column to isolate proteins which bound to this column and presumably bound to H3K27ac. Mass spectroscopy analysis of our CO-IP sample detected 763 proteins, many of which were not nuclear. We hypothesized that nonspecific binding of proteins to the antibody was to blame and determined to produce a nuclear extract in order to reduce nonspecific binding. We used a novel protocol produced by ChatGPT to partly purify nuclei from Tetrahymena using a short detergent extraction. These nuclei were then fully extracted and subjected to CO-IP. We subjected our sample to SDS-PAGE, which showed a great reduction in the number of proteins staining when compared to our whole cell extract sample. However, we still obtained multiple bands on our western blot, suggesting that non-specific binding of our antibody to proteins other than histone H3 may be occurring. Further research may involve changing the blocking conditions on our Western blot or finding a more specific antibody.
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.
Publication Date
2024
Nuclear Isolation Reduces Nonspecific Binding by anti-H3K27Ac Antibody in CO-IP Studies
Histone acetylation is critical for gene expression in eukaryotes. Acetylated lysines on histone tails are bound by bromodomain containing proteins which recruit chromatin remodeling proteins, site-specific transcription factors and other transcriptional apparatus. Our lab is particularly interested in the interactions between histone H3 acetylated at lysine 27 (H3K27ac) and any proteins that might be binding to it. In our previous work, we immunoprecipitated (using CO-IP) whole Tetrahymena extract with an anti-H3K27ac antibody and used an antibody binding column to isolate proteins which bound to this column and presumably bound to H3K27ac. Mass spectroscopy analysis of our CO-IP sample detected 763 proteins, many of which were not nuclear. We hypothesized that nonspecific binding of proteins to the antibody was to blame and determined to produce a nuclear extract in order to reduce nonspecific binding. We used a novel protocol produced by ChatGPT to partly purify nuclei from Tetrahymena using a short detergent extraction. These nuclei were then fully extracted and subjected to CO-IP. We subjected our sample to SDS-PAGE, which showed a great reduction in the number of proteins staining when compared to our whole cell extract sample. However, we still obtained multiple bands on our western blot, suggesting that non-specific binding of our antibody to proteins other than histone H3 may be occurring. Further research may involve changing the blocking conditions on our Western blot or finding a more specific antibody.
