![]() In some experiments, you might want to isolate your protein under native (non-denaturing) conditions. Other appropriate choices for fusion tags for Western blotting include 6XHis, C-Myc, HA, and HaloTag®. The FLAG® tag is a commonly-used tag for Western detection, due to its small size, and a prevalence of many good commercial antibodies for it. If you need to use a Western blot to detect expression of your protein from cell lysates, then choose a small tag for which there is a good antibody. If solubility is an issue, many researchers use the larger tags glutathione S-transferase (GST) or maltose binding protein (MBP). Instead, many researchers use another small tag, FLAG® (D-Y-K-D-D-D-D-K) for this purpose. coli, but it is not recommended for use in purification of non-secreted proteins from mammalian cells, due to a naturally high background of histidine. The 6XHis tag is widely used for purification from E. Typically, the small 6XHis tag (six histidine residues) is used for purification of proteins from cells. The tagged protein is eluted from the column in a highly purified form. Bacterial lysates are run through an affinity column, which binds to the tagged protein. One of the most common uses for tagging is protein purification, in which the tagged protein is usually expressed in E. However, if you require a tag, then the choice of tag will depend heavily on the application. A chief advantage of untagged ORFs is that they provide the opportunity to study the protein with its native structure. While GeneCopoeia ORF clones come with a choice of more than 150 tags and tag combinations (Table 1), they can also be provided in untagged format. Regardless of vector type, promoter, or tag, all GeneCopoeia OmicsLink™ Expression-ready ORFs are sequence-verified, and guaranteed to be free of PCR or cloning errors, frameshifts, premature stop codons, and single nucleotide polymorphisms (SNPs) not known to occur in nature. ORFs come either untagged, for expression of the protein with its native structure, or with N- or C-terminal fusion tags.Strong termination signals are added at the 3’ end, such as the SV40 polyA.Expression is placed under control of various promoters, such as CMV, EF1, etc.In these vectors, ORFs acquire the following features: GeneCopoeia strategy for transferring ORF sequences from full-length cDNAs to ready-to-use expression vectors lacking the natural 5’ and 3’ UTRs. The end result is that the natural 5’ and 3’ untranslated regions (UTRs) have been removed (Figure 1).įigure 1. ORFs are transferred to vectors from cDNAs, beginning with the initiator ATG and ending at the stop codon (the stop codon is removed when the ORF is connected to a C-terminal tag). GeneCopoeia’s OmicsLink™ Expression-ready ORF clones carry human and mouse ORF sequences. GeneCopoeia OmicsLink™ Expression-ready ORF clones for studying protein function In this Technical Note, we discuss the many tag choices available for GeneCopoeia ORF clones, and what you need to consider in order to make the best tag choice. However, designing a tagging strategy requires consideration of many factors, depending on the particular application and the goals of the experiment. These clones come either without tags or with a large number of fusion tag options for customers to choose from. GeneCopoeia provides tens of thousands of human and mouse open reading frame (ORF) clones for ready expression in vitro and in vivo. Fusion tagging-the practice of adding amino acids in-frame to one end of a native protein-is a well- established strategy for many applications, including protein purification, immunoprecipitation, Western blotting, and in vivo imaging. ![]()
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