Optimizing Biotin and V5 Tags for ChIP Assays
Author Information
Author(s): Kolodziej Katarzyna E, Pourfarzad Farzin, de Boer Ernie, Krpic Sanja, Grosveld Frank, Strouboulis John
Primary Institution: Erasmus MC
Hypothesis
The study aims to optimize the use of biotinylation tagging in chromatin immunoprecipitation (ChIP) assays to improve the reliability and efficiency of protein-DNA interactions.
Conclusion
The combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes.
Supporting Evidence
- The study demonstrates that omitting SDS during sonication improves ChIP enrichments.
- Using fish skin gelatin as a blocking agent significantly reduces background binding.
- The V5 epitope tag performs well under conditions optimized for streptavidin ChIP.
Takeaway
This study shows how using special tags can help scientists better see how proteins stick to DNA, which is important for understanding how genes work.
Methodology
The study involved optimizing conditions for streptavidin-ChIP using crosslinked chromatin from cells expressing a biotin-tagged transcription factor, including pre-clearing chromatin and blocking streptavidin beads.
Limitations
The study does not address the potential background binding due to naturally biotinylated histones, which could affect results.
Digital Object Identifier (DOI)
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