All measurements were normalized to reference DNA, a non-related sequence fragment amplified by PCR from gDNA, and spiked at 30 ng/sample before sonication

All measurements were normalized to reference DNA, a non-related sequence fragment amplified by PCR from gDNA, and spiked at 30 ng/sample before sonication. is Thiomyristoyl usually a wide variation in the design of different studies. One of the Thiomyristoyl most critical determinants of a successful ChIP-based approach is the antibody (5,11,15,16). ChIP antibodies should be capable of capturing specifically one single protein of a vast pool of DNA-binding proteins. It should also be considered that DNA binding and DNACprotein cross-linking might provoke conformational changes in the nucleoprotein complexes that lead to epitope masking, causing false-negative outcomes, whereas cross-reactivity of the antibodies to non-cognate targets could generate false-positive outcomes. Effects of epitope masking can be minimized by using polyclonal antibodies (pAbs) (17). However, pAbs increase the frequency of false-positive outcomes, their production requires regular immunization and they exhibit batch to batch variability (18,19). In comparison with pAbs, monoclonal antibodies (mAbs) suffer less from the aforementioned problems. However, the availability of high-quality ChIP-grade mAbs is usually apparently limited (11,20). Epitope tagging, by homologous recombination-mediated knock-in of the tagged genes, could circumvent the lack of ChIP-grade mAbs. Although this technology is usually relatively straightforward for some well-established model organisms, such as and (7,8,14,21C23), genetic tools to achieve this in many organisms such as during immunization with antigen. As camelid heavy-chain antibodies bind their target antigens by only Thiomyristoyl one single domain, construction of large immune libraries to trap antigen-specific nanobodies? has proven unnecessary (27,28). Construction of libraries of antigen-binding repertoire of conventional antibodies is also complicated by the presence of multiple VH and VL gene families, whereas the vast majority of VHHs belong to one single sub-family (28). The aforementioned technological advantages of constructing immune nanobody? libraries, together with small size, recognition of unique epitopes, high affinity, high solubility, high expression yield in heterologous expression systems and easy tailoring, make nanobodies? an interesting class of affinity reagents for various applications (27,29,30). Here, we demonstrate the use of target-specific nanobodies? in ChIP experiments. As a model system, we chose the well-characterized transcription regulator Ss-LrpB from the hyperthermoacidophilic archaeon (31). Ss-LrpB belongs to the leucine-responsive regulatory protein (Lrp) family, a widespread and abundant family of regulators in prokaryotes, both bacteria and archaea (32,33). Several regulatory targets of Ss-LrpB have already been identified by binding experiments and by gene expression analysis (34). These targets include the regulator gene itself and a gene cluster juxtaposed to it, encoding a putative ferredoxin oxidoreductase and two permeases. In this work, different Ss-LrpB-specific nanobodies? were generated and assessed for their capacity to capture specifically the regulator, either free or bound to DNA. We then developed a nanobody? -based ChIP protocol for and was purified by heat treatment and ion exchange chromatography, as previously described (35). The His-tagged C-terminal domain name of Ss-LrpB was purified by Ni2+ affinity chromatography (36). LysM and Ss-Lrp proteins were produced and purified by the same procedure as the Ss-LrpB purification. For LysM, BL21(DE3) was first transformed with construct pLUW632 (37). After purification, the Ss-LrpB and Ss-Lrp preparations were dialysed against 20 mM of TrisCHCl (pH 8.0), 50 mM of NaCl, 0.4 mM of ethylenediaminetetraacetic acid (EDTA), 0.1 mM of DTT, 12.5% of glycerol and the LysM preparation against 20 mM of TrisCHCl (pH 8.0) and 20% of glycerol. After identification as described CLTB later in the text, the Ss-LrpB-specific VHH (nanobody?) genes were cloned into the pHEN6c vector, which allows expression of nanobodies? in fusion with His6 tag (38). Expression and purification of nanobodies? were performed as previously described (39). Protein concentrations in the case of Ss-LrpB expressed in monomeric units were determined by ultraviolet absorption at 280 nm and by densitometric analysis of Coomassie stained sodium dodecyl sulphate (SDS)Cpolyacrylamide gel (PAG). Generation of Ss-LrpB-specific nanobodies? Ss-LrpB-specific nanobodies? were generated by immunizing an alpaca (BL21(DE3) crude cell extracts containing one of the three Lrp-like transcription factors from (Ss-LrpB, LysM Thiomyristoyl or Ss-Lrp), expressed from recombinant pET24 vectors, were used for these experiments. Crude extracts from BL21(DE3) made up of an empty pET24 vector served as unfavorable control. Cell pellets from 20 ml cultures were resuspended in 1 ml of IP buffer [150 mM of NaCl, 50 mM of TrisCHCl (pH 8.0), 1% of Triton X-100, 0.5% of NP-40, 1% of deoxycholate], sonicated and centrifuged. Aliquots of 200 l of the supernatants were incubated with.

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