Syndicated from PubMed RSS Feeds
Nanobody-based chromatin immunoprecipitation.
Methods Mol Biol. 2012;911:491-505
Authors: Duc TN, Hassanzadeh-Ghassabeh G, Saerens D, Peeters E, Charlier D, Muyldermans S
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.
PMID: 22886272 [PubMed - in process]
More...
Nanobody-based chromatin immunoprecipitation.
Methods Mol Biol. 2012;911:491-505
Authors: Duc TN, Hassanzadeh-Ghassabeh G, Saerens D, Peeters E, Charlier D, Muyldermans S
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.
PMID: 22886272 [PubMed - in process]
More...