Abstract
Chromatin immunoprecipitation (ChIP), followed by microarray hybridization (ChIP-chip) or high-throughput sequencing (ChIP-seq), is becoming a widely used powerful method for the analysis of the in vivo DNA–protein interactions at genomic scale.
The success of ChIP largely depends on the quality of antibodies. Although polyclonal antibodies have been successfully used for ChIP, their production requires regular immunization and they exhibit high aspecificity and batch to batch variability. These problems can be circumvented by generating monoclonal antibodies (mAbs) via hybridoma technology. However, such mAbs do not often capture DNA–protein complexes and are not amenable to engineering.
Nanobodies are recombinant single domain antibody fragments derived from camelid Heavy-Chain antibodies. Nanobodies exhibit high affinity and specificity towards their cognate antigens and often capture their target antigens in solution. Moreover, the Nanobody genes can be easily tailored to streamline ChIP.
Here, we describe a Nanobody-based ChIP protocol which we have successfully used for genome-wide identification of the binding sites of the low-abundant transcription factor Ss-LrpB from the hyperthermoacidophilic archaeon Sulfolobus solfataricus.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Grogan DW (2000) The question of DNA repair in hyperthermophilic archaea. Trends Microbiol 8:180–185
She Q et al (2001) The complete genome of the crenarchaeon Sulfolobus solfataricus P2. Proc Natl Acad Sci USA 98:7835–7840
Orlando V (2000) Mapping chromosomal proteins in vivo by formaldehyde-crosslinked chromatin immunoprecipitation. Trends Biochem Sci 25:99–104
Orlando V, Strutt H, Paro R (1997) Analysis of chromatin structure by in vivo formaldehyde cross-linking. Methods 11:205–214
Nelson JD, Denisenko O, Bomsztyk K (2006) Protocol for the fast chromatin immunoprecipitation (ChIP) method. Nat Protoc 1:179–185
Haring M et al (2007) Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods 3:11
Weinmann AS, Farnham PJ (2002) Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation. Methods 26:37–47
Dahl JA, Collas P (2007) Q2ChIP, a quick and quantitative chromatin immunoprecipitation assay, unravels epigenetic dynamics of developmentally regulated genes in human carcinoma cells. Stem Cells 25:1037–1046
Mukhopadhyay A et al (2008) Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans. Nat Protoc 3:698–709
Wells J, Farnham PJ (2002) Characterizing transcription factor binding sites using formaldehyde crosslinking and immunoprecipitation. Methods 26:48–56
Carroll JS et al (2006) Genome-wide analysis of estrogen receptor binding sites. Nat Genet 38:1289–1297
Impey S et al (2004) Defining the CREB regulon: a genome-wide analysis of transcription factor regulatory regions. Cell 119:1041–1054
Robertson G et al (2007) Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nat Methods 4:651–657
Peeters E et al (2004) Ss-LrpB, a novel Lrp-like regulator of Sulfolobus solfataricus P2, binds cooperatively to three conserved targets in its own control region. Mol Microbiol 54:321–336
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-[Delta][Delta]CT method. Methods 25:402–408
Fan X et al (2008) Extensive chromatin fragmentation improves enrichment of protein binding sites in chromatin immunoprecipitation experiments. Nucl Acids Res 36:e125
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Duc, T.N., Hassanzadeh-Ghassabeh, G., Saerens, D., Peeters, E., Charlier, D., Muyldermans, S. (2012). Nanobody-Based Chromatin Immunoprecipitation. In: Saerens, D., Muyldermans, S. (eds) Single Domain Antibodies. Methods in Molecular Biology, vol 911. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-968-6_31
Download citation
DOI: https://doi.org/10.1007/978-1-61779-968-6_31
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-967-9
Online ISBN: 978-1-61779-968-6
eBook Packages: Springer Protocols