Co-IP using an antibody versus pull-down assay using Strep-Tactin®XT
Whereas IP and Co-IP utilize immobilized antibodies to purify the bait protein along with its binding partners (prey), in the latter case from cell lysates for analysis of the whole protein complex, in pull-downs the target is fused to an affinity tag and captured together with bound proteins using a corresponding immobilized chemical or biological ligand. In every case, the received sample is further analyzed by e.g., SDS-PAGE, western blot, or mass spectrometry.
The advantage of pull-down over IP and Co-IP are mild elution conditions. They retain the natural protein structure as well as protein complexes and prevent elution of unspecific bound proteins leading to robust results in subsequent analytic applications. This is especially the case for the Strep-tag® technology.
For pull-down assays, the Strep-tag® technology is compatible with magnetic and non-magnetic experimental setups as well as any protein class and offers versatile resins for the realization of a multitude of assays to detect interactions. For small amounts of a sample, MagStrep “type3” XT beads can be chosen or when up-scaling of the process is anticipated, agarose-based resins display a powerful option.
Alternatively, for IP and Co-IP, IBA provides selector resins with a high-affinity single-domain antibody (sdAb) either for GFP, GST, MBP, RFP or TagFP-tagged fusion proteins. These selector resins show negligible non-specific background after precipitation and GFP and RFP Selector resins are applicable for a wide range of their derivatives as well.
Conventional IP using antibodies versus IP with Selector Resins
Capturing Twin-Strep-tag® proteins with BiacoreTM
A crucial step in SPR analysis is the immobilization of the ligand to the surface of the biosensor chip. The immobilization of the ligand can take place via direct covalent coupling to the surface of the chip or by capture with the help of a pre-coated capture molecule. However, the direct covalent immobilization of the ligand can change its biological activity or result in an undirected immobilization with reduced accessibility of the binding sites. The transient immobilization of tagged ligands via affinity capturing molecules overcomes these drawbacks.
Nevertheless, tags used for such applications must bind efficiently to the capturing molecule with an affinity that is higher than that between analyte and ligand. His-tagged ligands that are immobilized on an NTA-surface with a KD in the nanomolar range can be used for measuring weaker interactions, but they fail in the measurement of high-affinity interactions with slow dissociation rates. In contrast, Strep-Tactin®XT forms an exceptionally strong complex with the Twin-Strep-tag®. An affinity in the low picomolar range allows measurements of long dissociation times and slow off-rates.
Thus, the combination of these two components of the Strep-tag® technology enables a directed, high-affinity immobilization that does not influence the activity of the ligand or require any modifications. Due to the high-affinity interaction and its high specificity, non-specific binding of host cell proteins is avoided, and ligands can be captured efficiently and directly from culture media. Moreover, Strep-Tactin®XT is compatible with many substances and the biosensors can be easily regenerated. The Twin-Strep-tag® Capture Kit provides all necessary products for the preparation of Strep-Tactin®XT-coated biosensor chips and the following measurements.