We developed low affinity Fab fragments or selected nanobodies that weakly bind to the cell. Only an increase in avidity (i.e. combining the individual binding strengths) ensures that cells are efficiently captured. This is achieved by multimerizing Fab fragments or nanobodies on a backbone such as Strep-Tactin®, which is a streptavidin derivative.
Strep-Tactin® is, like streptavidin, a tetrameric molecule with four biotin binding pockets. We developed an amino acid sequence that binds to two of these pockets. This amino acid sequence is called Twin-Strep-tag® and is fused as a tag to our Fab fragments (Fab-Streps) or nanobodies (Nano-Streps).
Strep-Tactin® is available as different conjugates that are all suitable for traceless affinity cell selection (TACS). For fluorescent cell staining and sorting, we offer Strep-Tactin® conjugated to fluorophores PE or APC. We also offer columns filled with an agarose coupled to Strep-Tactin® (Strep-Tactin® TACS Agarose) that can be used for selecting cells using affinity chromatography. Strep-Tactin® Magnetic Microbeads are suitable for magnetic labeling and isolation of cells.
Although Strep-Tactin® efficiently binds to the Twin-Strep-tag® and thereby immobilizes Fab fragments or nanobodies, it has a much higher affinity to biotin. Addition of 1 mM biotin causes the complete release of all bound Fab fragments or nanobodies. At the same time, the multimerization is dissolved and Fab fragments/nanobodies spontaneously dissociate from the cells due to their low affinity. This way, the eluted cells are completely label-free.
Here are some example data for CD3+ T cell isolation from murine splenocytes (n = 7) using affinity chromatographic Fab-TACS®:
CD3+ cells were isolated from murine splenocytes (A, n=7) using the affinity chromatographic Fab- TACS® isolation method. Data are displayed as mean ± SEM. Cells were analyzed by flow cytometry. Representative FACS plots before and after enrichment are shown in (B). An average of 91% CD3+ T cells demonstrates that highly pure populations could be isolated. A yield of > 85% (C) also shows that the isolation procedure is very efficient. In addition, a viability of > 91% (D) highlights that this method is stress-free for cells.
Low affinity Fab fragment fused to a Twin-Strep-tag® and specific for murine CD3
Low affinity Fab fragment fused to a Twin-Strep-tag® and specific for murine CD4
Low affinity Fab fragment fused to a Twin-Strep-tag® and specific for murine CD3
Low affinity Fab fragment fused to a Twin-Strep-tag® and specific for murine CD4
Strep-Tactin® multimers conjugated with magnetic microbeads
Gravity flow column suitable for traceless affinity cell and exosome isolation
100 mM concentrated biotin for dissociation of Twin-Strep-tag® from Strep-Tactin® suitable for cells
Label-free isolation of T cells from murine splenocytes
Label-free isolation of T helper cells from murine splenocytes
Label-free isolation of cytotoxic T cells from murine splenocytes
Label-free isolation of B cells from murine splenocytes
Use your own capture proteins (Fab fragments, nanobodies or antibodies) and combine them with our Strep-Tactin® products for cell isolation!
Requirement: Capture proteins should have a Twin-Strep-tag®. Desthiobiotinylated or biotinylated (depletion only!) capture proteins are also an option.