Spot-Tag® peptide sequence is derived from the protein beta-catenin and its sequence has been optimized for higher affinity binding to the Spot-Tag Nanobody (Figure 1). Beta-catenin background, i.e. binding of beta-catenin to the Spot-Tag Nanobody is rated negligible.
In detail, Spot-Tag has been engineered from a linear epitope from the unstructured N-terminus (aa 16-27) of beta-catenin. This sequence is called BC2. The wild-type sequence has been affinity optimized by off-rate screenings (Figure 2). As a result, Spot-tagged proteins are preferentially bound by the Spot-Nanobody, because Spot-Nanobody has a one order of magnitude better affinity to Spot-Tag than to the wild-type sequence BC2 (Table 1). Spot-Trap binds to the Spot-Tag peptide with a dissociation constant of just 0.7 nM.
Table 1: Kinetic data
Dissociation constant KD, on-rates and off-rates of a N-terminally tagged Spot-fusion protein in comparison to the corresponding N-terminally tagged BC2-tag-fusion protein quantitate that the Spot-Nanobody has an about 10-fold higher affinity to Spot-tag.
Immunoprecipitation and affinity purification
In the presence of a Spot-tagged protein, the beta-catenin background of the Spot-Trap is negligible. This is because the anti-Spot-tag VHH has a significantly higher affinity to the Spot-tag peptide than to wildtype beta-catenin (BC2). Hence, Spot-tagged proteins are better bound by Spot-Trap. Furthermore, expression levels of heterologously expressed Spot-tagged proteins are usually much higher than endogenous beta-catenin-levels.
However, for very sensitive methods such as Mass Spectrometry, binding to beta-catenin-binding may be still detectable, as well as some of the beta-catenin interaction partners such as alpha-catenin. Since these are known contaminants, these peptides can be easily subtracted during data analysis or used for alignments of control and sample. Therefore, this doesn’t cause any problems; as always, appropriate background controls, e.g. for immunoprecipitations using mock lysate, are highly recommended.
Finally, our data indicate that Spot-Trap only binds to beta-catenin, if this epitope is dephosphorylated, which reduces background levels even further.
Virant et al have thoroughly assessed the background staining of endogenous beta-catenin by the Spot-Nanobody, called BC2-Nanobody in the publication "Nature Communications" (2018) doi:10.1038/s41467-018-03191-2. The authors conclude that the BC2-epitope of wild type beta-catenin (BC2Tag) has only a minor impact on immunostaining of BC2-tagged proteins. Due to its superior binding to the Spot-Tag, the Spot-Tag Nanobody causes even less beta-catenin-derived background staining when working with Spot-tagged proteins. This is strongly supported by ChromoTek’s imaging data, in which no background from endogenous beta-catenin was observed.
Spot-Trap is a very low background affinity capture reagent
Spot-Trap has been benchmarked with commonly used affinity reagents from other suppliers (Figure 3). Here, Immunoprecipitations (IP) were conducted from HEK293T cell lysates without Spot-Tag present; experiments were normalized for equal binding capacity. The IPs were done according to respective suppliers’ protocols:
As a result, the Spot-Trap clearly has the lowest background of all tested products. This makes Spot-Trap a superior affinity capture tool for single band purification of Spot-tagged proteins.