Skip to main content
. Author manuscript; available in PMC: 2019 Jan 4.
Published in final edited form as: Methods Mol Biol. 2019;1891:37–49. doi: 10.1007/978-1-4939-8904-1_5

Fig. 2.

Fig. 2

Example of a multi-reaction cycle. (a) Three different receptor-Fc fusion proteins are captured sequentially on a four-channel sensor chip (R1-Fc, R2-Fc, and R3-Fc). Capture is highly reproducible as seen in the exact matching of ten capture cycles for R1-Fc (from approximately 200 to 300 s), R2-Fc (from approximately 550 to 650 s), and R3-Fc (from approximately 900 to 1000 s). (b) BMP at a concentration of 80 nM is injected over the sensor chip following receptor capture. In the example shown here, 15 different members of the TGF-β/BMP family were injected to test for binding to BMPRII-Fc. Each curve represents a different molecule. Each protein elicits a specific response as seen in the distinct association rates (from 0 to 300 s) and dissociation rates (from 300 to 1100 s). The strongest BMPRII-Fc binders were activin B (black response curve of approximately 110 RU) and nodal (dark gray response curve of approximately 100 RU). (c) Regeneration restores the baseline response ±5 RU (from 900 to 1000 s) by eluting the captured receptor-Fc fusion protein, including the receptor-bound protein. Regeneration conditions are highly reproducible with this approach as seen in this superposition of ten growth factor-binding/regeneration cycles. Regeneration steps are labeled as in the following panel. (d) In this close-up view of the sensor chip regeneration, the residual binding response (BMP, from 0 to 100 s) can be observed at the beginning of the regeneration cycle. Magnesium chloride (MgCl2, from 100 to about 450 s) elutes bound receptors and associated ligands. Re-equilibration with buffer (HBS, from about 450 to about 650 s) restores the baseline. A washing step with water (H2O, from about 650 to about 825 s) is necessary to avoid magnesium chloride accumulation and clogging of the microfluidics unit. A second equilibration with buffer (HBS, from about 825 to about 1000 s) completes the regeneration program, and the sensor chip returns to a constant RU baseline