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Scrubber2

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The SCRUBBER2 is a laboratory equipment designed to remove unwanted gases or vapors from a stream of air or other gases. It utilizes a scrubbing mechanism to purify the incoming gas stream.

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Lab products found in correlation

Biacore t200 machine, by GE Healthcare (3 mentions) Prism version 8, by GraphPad (2 mentions)

3 protocols using scrubber2

1

Surface Plasmon Resonance of PTCH1-ShhN Binding

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SPR experiments were performed using a BIAcore T200 machine (GE Healthcare) at 25°C in 10 mM HEPES, pH 7.5, 150 mM NaCl, 3 mM CaCl2, 0.005% (v/v) Polysorbate 20. The biotinylated PTCH1ECD1-ECD2 was immobilized via streptavidin onto a CM5 sensor Chip. PTCH1ECD1-ECD2 surface concentration was 820 response units. ShhNC24II (used as analyte) was dialyzed against SPR running buffer prior to use and a two-fold dilution series was prepared. The experiment was run at 5 µl/min with 600 s association and 400 s dissociation. The signal from the experimental flow cell was corrected by subtraction of a buffer and the reference signal from a control flow cell (streptavidin coated). All data were analyzed using SCRUBBER2 (Biologic) and GraphPad Prism Version 8 (GraphPad Software, San Diego USA). The dissociation constant (Kd) was obtained by nonlinear regression using a “one-site specific binding” model (Y=Bmax∙X/(Kd+X), where X is analyte concentration and Bmax is the maximum analyte binding).
Rudolf A.F., Kinnebrew M., Kowatsch C., Ansell T.B., El Omari K., Bishop B., Pardon E., Schwab R.A., Malinauskas T., Qian M., Duman R., Covey D.F., Steyaert J., Wagner A., Sansom M.S., Rohatgi R, & Siebold C. (2019). The morphogen Sonic hedgehog inhibits its receptor Patched by a pincer grasp mechanism. Nature chemical biology, 15(10), 975-982.
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2

Membrane Protein Binding Kinetics

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We performed equilibrium experiments using a Biacore T200 machine (GE Healthcare) at 25°C. The experiments were carried out at pH 7.5 (PBS, 0.005% [v/v] polysorbate 20), unless indicated otherwise. Experiments at pH 5.7 were run in 150 mM NaCl and 50 mM citric acid. The regeneration buffer was 2 cM MgCl2. To mimic the native membrane insertion topology, we biotinylated proteins enzymatically at the C-terminal avidity tag and attached the resulting biotin label to streptavidin-coated Biacore chip surfaces. Data were analyzed with Scrubber2 (BioLogic). Kd and maximum analyte binding (Bmax) values were obtained by nonlinear curve fitting of a 1:1 Langmuir interaction model (bound = Bmax/(Kdc+cC), where C is analyte concentration calculated as monomer).
Seiradake E., del Toro D., Nagel D., Cop F., Härtl R., Ruff T., Seyit-Bremer G., Harlos K., Border E.C., Acker-Palmer A., Jones E.Y, & Klein R. (2014). FLRT Structure: Balancing Repulsion and Cell Adhesion in Cortical and Vascular Development. Neuron, 84(2), 370-385.
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3

SPR Analysis of PTCH1-ShhN Interaction

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SPR experiments were performed using a BIAcore T200 machine (GE Healthcare) at 25 °C in 10 mM HEPES, pH 7.5, 150 mM NaCl, 3 mM CaCl2, 0.005% (v/v) Polysorbate 20. The biotinylated PTCH1ECD1-ECD2 was immobilized via streptavidin onto a CM5 sensor Chip. PTCH1ECD1-ECD2 surface concentration was 820 response units. ShhNC24II (used as analyte) was dialyzed against SPR running buffer prior to use and a two-fold dilution series was prepared. The experiment was run at 5 μl/min with 600 s association and 400 s dissociation. The signal from the experimental flow cell was corrected by subtraction of a buffer and the reference signal from a control flow cell (streptavidin coated). All data were analyzed using SCRUBBER2 (Biologic) and GraphPad Prism Version 8 (GraphPad Software, San Diego USA). The dissociation constant (Kd) was obtained by nonlinear regression using a “one-site specific binding” model (Y=Bmax∙X/(Kd+X), where X is analyte concentration and Bmax is the maximum analyte binding).
Rudolf A.F., Kinnebrew M., Kowatsch C., Ansell T.B., El Omari K., Bishop B., Pardon E., Schwab R.A., Malinauskas T., Qian M., Duman R., Covey D.F., Steyaert J., Wagner A., Sansom M.S., Rohatgi R, & Siebold C. (2019). The morphogen Sonic hedgehog inhibits its receptor Patched by a pincer grasp mechanism. Nature chemical biology, 15(10), 975-982.
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