Strep-tag® technology for cell isolation
The Strep-tag® technology provides methods for isolating cells via their surface markers (Traceless Affinity Cell Selection - Fab-TACS®/Nano-TACS®) or via their antigen specificity (MHC I Streptamer®).
The Twin-Strep-tag® facilitates the binding of either Fab- or Nano-Streps to Strep-Tactin®, an engineered streptavidin. Different Strep-Tactin® conjugates are the basis for the three cell isolation approaches. For affinity chromatography, Fab- /Nano- Streps of choice are loaded into Strep-Tactin® TACS Agarose-containing columns. For magnetic cell isolation, Fab-/Nano-Streps are bound to Strep-Tactin® conjugated to magentic microbeads, whereas combining them with fluorophore-conjugated Strep-Tactin® allows fluorescent cell sorting.
Find application examples of Fab-TACS® affinity chromatographic isolation and Fab-TACS® magnetic isolation here.
Fab-TACS®/Nano-TACS® cell isolation methods
In the Fab-TACS®/Nano-TACS® approach, low affinity Fab-Streps or nanobodies against specific surface markers are multimerized on different Strep-Tactin® backbones to facilitate three isolation methods: affinity chromatographic isolation using TACS Agarose, magnetic isolation using Strep-Tactin® conjugated to magnetic microbeads and fluorescent cell sorting using Strep-Tactin® conjugated to fluorophores.
The key feature of the Fab-TACS®/Nano-TACS® principle is the removability of all labeling reagents. Two factors account for this feature: Low affinity Fab-/Nano-Streps and the reversible binding of the Twin-Strep-tag® to Strep-Tactin®.
The Fab fragments and nanobodies bind specifically, but only weakly, to the target. To achieve sufficient binding to the cells, Fab-/Nano-Streps are multimerized on Strep-Tactin® molecules to increase the avidity. Biotin addition reverses this binding. Due to its higher affinity, biotin replaces the Twin-Strep-Tag® on Strep-Tactin® molecules and thereby causes the release of Fab-/Nano-Streps. Due to their low affinity, Fab-/Nano-Streps spontaneously dissociate from the cell surface after being monomerized, resulting in a completely label-free cell population. Cells are only minimally affected during the isolation procedures and suitable for various downstream applications.
Multimerization increases the avidity
Due to their low affinity, a multimerization of Fab-Streps is required to increase the avidity sufficiently to immobilize target cells. This is achieved by using agarose beads coated with several Strep-Tactin® molecules that are filled into a column, by using Strep-Tactin® multimers conjugated to magnetic microbeads or by cross-linking Strep-Tactin® molecules with large fluorophores.
Reversible reagents for label-free cells
All Fab-TACS®/Nano-TACS® cell isolation methods use reversible reagents to generate label-free cells. Adding biotin releases the Fab-Streps from Strep-Tactin® and causes their spontaneous dissociation from the cells due to their low affinity as monomers.
In the Fab-TACS® approach that uses affinity chromatography as underlying isolation method, Fab-Streps of choice are loaded into columns containing an agarose coated with Strep-Tactin® that is suitable for traceless affinity cell selection (TACS). After the sample is applied, unwanted material is washed away. Finally, cells are eluted/released by adding biotin. Due to the low affinity of the Fab-Streps, they spontaneously dissociate from their target, yielding completely label-free cells.
Fab-TACS® affinity chromatographic cell isolation workflow
The key feature of MHC I Streptamer® isolation is the same as for Fab-/Nano-TACS®: the possibility to remove all labeling reagents after successful isolation. Low affinity of monomeric MHC I-peptide complexes to the T cell receptor and the reversible binding of the Twin-Strep-tag® to Strep-Tactin® are two factors that contribute to this feature. Biotin addition initiates the dissociation of all reagents from the cells. Due to its higher affinity, biotin replaces the Twin-Strep-Tag® on Strep-Tactin® molecules and thus causes the release of MHC I-Streps.
This reverses the multimerization of MHC I-peptide complexes and leads to their spontaneous dissociation from the cell surface due to their low affinity as monomers. The resulting cell population is completely label-free. This keeps the effects of the isolation procedure on the cells to a minimum, making them suitable for various downstream applications. The benefit of label-free cells has been shown in multiple scientific publications.
The following application note demonstrates how MHC I Streptamer® and Fab-TACS® approaches can be combined for tandem cell isolation.
MHC I Streptamer® magnetic cell isolation workflow
The MHC I Streptamer® magnetic cell isolation workflow includes the following steps: MHC I-Streps have to be loaded on Strep-Tactin® Magnetic Microbeads. The resulting complexes are used to label cells for subsequent positive selection on a magnet. After washing away all unwanted cells, biotin addition releases MHC I-Streps from Strep-Tactin® Magnetic Microbeads and causes the spontaneous dissociation of MHC I-Streps from the cell surface (elution).
Fab-TACS® cell staining workflow
The Fab-TACS® cell staining workflow includes the following steps: Fab-Streps have to be loaded on fluorescently conjugated Strep-Tactin®. The resulting complexes are used to labels cells for subsequent flow cytometric analysis and/or cell sorting. After washing away all unwanted cells, biotin addition releases Fab-Streps from fluorescent Strep-Tactin® and causes the spontaneous dissociation of Fab-Streps from the cell surface (elution).
T cell stimulation & expansion workflow using CD3/CD28 Streptamers
T cell stimulation & expansion using CD3/CD28 Streptamers includes the following steps: combining CD3- and CD28-Fab Streps with Strep-Tactin® multimers, adding the soluble CD3/CD28 Streptamer® complexes to cells for activation and expansion and terminating the stimulation by adding biotin.
Find out more about Strep-tag® cell isolation
Here, we have grouped links to make it easier to find more information about Strep-tag® technology for cell isolation. Find the products that fit your research needs. Have a look at the frequently asked questions about the use of Strep-tag® for cell isolation and references. Find application notes, brochures and flyers in our download area and browse through the application examples.