Optimal use of tandem biotin and V5 tags in ChIP assays
1 Department of Cell Biology, Erasmus MC, Dr Molewaterplein 50, 3015GE Rotterdam, the Netherlands
2 Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands
3 Institute of Molecular Oncology, Biomedical Sciences Research Center Alexander Fleming, PO Box 74145, 16602 Varkiza, Greece
BMC Molecular Biology 2009, 10:6 doi:10.1186/1471-2199-10-6Published: 5 February 2009
Chromatin 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.
Using 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.
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.