In fact, a tag initiating in S12 (tag 12C24) resulted in a pattern of bands equally recognized by anti-SV5 and 1F2, both in the intracellular and in the secreted material (Figure 4D, lanes 1C4), indicating that in the absence of P11, O-glycosylation was not taking place, while N-glycosylation was still present

In fact, a tag initiating in S12 (tag 12C24) resulted in a pattern of bands equally recognized by anti-SV5 and 1F2, both in the intracellular and in the secreted material (Figure 4D, lanes 1C4), indicating that in the absence of P11, O-glycosylation was not taking place, while N-glycosylation was still present. detection of several recombinant cytosolic, secretory and membrane proteins. Two additional variants of roTag of 10 and 13 amino acids containing O-glycosylation susceptible sites (termed OG-tag and roTagO) were constructed and characterised. These tags were useful to detect proteins passing through the Golgi apparatus, the site of O-glycosylation. Introduction In biological sciences development of new specific monoclonal antibodies (mAbs) is a pressing requirement for several aspects in the field: from basic research on protein function, to medical diagnosis, prophylaxis and therapy of several pathogenic conditions [1], [2], [3], [4], [5], [6]. Taking advantage of the hybridoma technology to produce monoclonal antibodies of desired specificity [7], [8], a number of mAb/epitopes pairs derived from different proteins have been characterized and used as tags to facilitate identification of recombinant proteins. Indeed, epitope tagging is a common methodology used to identify recombinant proteins when specific antibodies for the protein of interest are not readily available [9]. This technique consists in the expression of fusion proteins, obtained by inserting a nucleotide sequence encoding a peptide tag into the gene of interest. Usually a peptide tag is a short cIAP1 Ligand-Linker Conjugates 14 peptidic sequence (an epitope) recognized by an already existing antibody [10]. Tags can be used for protein detection in immunoenzymatic or immunochemical assays, as well as for protein isolation and purification by immunoprecipitation or affinity cIAP1 Ligand-Linker Conjugates 14 chromatography [11], [12]. Epitope tagging can help in the characterization of the tagged protein, by facilitating the determination of its abundance, cellular location, post-translational modifications, interactions with other proteins, etc. In addition, if the tag-specific antibody displays differential affinity depending on different post-translational modifications (e.g. phosphorylation or glycosylation) on the tag sequence itself, this can be exploited, for instance, to obtain information about activation cIAP1 Ligand-Linker Conjugates 14 status [13] or trafficking of the tagged protein through cellular compartments where those modifications take place [14]. Epitope tagging offers a number of advantages over alternative detection and purification methods, because it saves time and resources comparing with the traditional methods for producing specific antibodies (either monoclonal or polyclonal) to the protein of interest. As tags are often short (6C15 amino acids in length), they are generally presumed to have no effect on the biological functions of the tagged proteins. However, if located in inappropriate positions, they might interfere with protein structure, function and interactions. In addition, not all mAb are suitable for every immunodetection method, as in the case of mAb specific for non-linear epitopes. For those reasons, it is useful to develop mAbs and epitope tags of different sequence characteristics (size, net charges, hydrophobicity and side groups) or that can be fused in different positions of the target protein to increase the chances of success in tagging applications. Here we describe and characterize a new 10 amino acids long epitope tag (roTag) derived from the sequence of the rotavirus (RV) non-structural protein 5 (NSP5). NSP5 has an essential role during the RV replication cycle, as it is essentially required for the assembly of viroplasms, the sites of viral genome replication and initial assembly of progeny virus [15], [16]. In this context, since the precise role of NSP5 is still poorly understood [17], [18], we developed a series of novel mAbs reacting with different NSP5 domains. One highly specific anti-NSP5 mAb (1F2/anti-roTag) was identified FSCN1 and the recognized cIAP1 Ligand-Linker Conjugates 14 minimal linear epitope was mapped. The epitope, termed roTag, was shown to be highly specific when fused to reporter proteins. Further variants of roTag have been derived, including an O-glycosylation site, that proved useful to determine whether proteins in the secretory pathway have trafficked through the Golgi, according to their O-glycosylation status. Results and Discussion Characterization of anti-roTag mAb A panel of anti-NSP5 mAbs were generated from BALB/c mice immunized with a Ni++-purified His-tagged NSP5 protein of the RV porcine OSU strain [19]. Screening of more than 400 clones by ELISA yielded 20 positive clones, of which 6 were further confirmed positive in RV-infected cells. mAb 1F2 (IgG1 cIAP1 Ligand-Linker Conjugates 14 isotype) was.