Abstract
BackgroundChromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition, we have employed protein in vivo biotinylation tagging as a very high affinity alternative to antibodies. In this paper we describe the optimization of biotinylation tagging for ChIP and its coupling to a known epitope tag in providing a reliable and efficient alternative to antibodies.ResultsUsing the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking.ConclusionThe 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.
Highlights
Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-onchip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins
We previously showed using biotin-tagged GATA-1 that streptavidin binding of crosslinked chromatin can substitute for antibodies in enriching for GATA-1 target genes in ChIP assays [7]
Due to the potential advantages offered by the very high affinity of streptavidin for binding to biotin and the importance of having multiple tags that can be used in the same cell, we wanted to extend these observations in developing an optimized protocol for the streptavidin binding of chromatin from cells expressing biotin-tagged GATA-1
Summary
Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-onchip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. Affinity tags have been widely used for the study of protein interactions and the isolation of protein complexes Such tags are increasingly used in ChIP assays in detecting the in vivo binding of transcription factors and associated co-factors to their target genes in chromatin. In searching for the optimal affinity tag for ChIP applications, three criteria are important: (a) tags must have high binding affinity; (b) tags should be preferably small and not strongly charged so as to minimize possible interference with transcription factor function (c) tags should be fairly insensitive to formaldehyde fixation. The latter is (page number not for citation purposes). Once formed, the biotin-streptavidin complex is not disturbed by changes in pH, introduction of detergents or high salt concentration, remaining stable even under very stringent washing conditions; (b) biotin is a very small molecule and is not known to affect the biological activity of tagged proteins [5,6]; (c) there are few (mostly cytoplasmic) naturally biotinylated proteins in mammalian cells, as a result the non-specific background binding of nuclear extract is low [7]
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