Alexa 647 nhs

Tension probes based on the I27 domain of titin were expressed in BL21 chemocompetent E. coli (#C2530H, NEB, USA) as previously described^{20 (link)} from the plasmid pET22b-I27-RGD/E for the modified I27 domains containing an RGD or an RGE sequence for specific integrin adhesion or no specific adhesion, respectively, and the amber codon (TAG) as well as the pEVOL-pAzF plasmid for p-azidophenylalanine incorporation at the amber codon. Proteins were purified by immobilized metal ion affinity chromatography with a 1 ml His-Trap column (Äkta pure system, GE healthcare, UK) with buffer A: KH₂PO₄ buffer (pH 7.4) + 1 mM DTT + 30 mM imidazole and a gradient of 0–100 % buffer B: KH₂PO₄ buffer (pH 7.4) + 1 mM DTT + 500 mM imidazole. Purified proteins were desalted by gel filtration with Zeba spin columns (7000 MWCO, Thermo Fischer Scientific, USA) and subsequently labeled with ten-fold molar excess of Alexa647-NHS (Thermo Fischer Scientific, USA) in 0.1 mM KH₂PO₄ buffer at RT overnight. Unbound dye was removed again by gel filtration. Protein and dye concentration were quantified by UV–Vis absorption (NanoDrop ND-1000, Peqlab, Germany). Degree of labeling was determined to be 1.2, hence the majority of titin-based tension probes bears 1 fluorophore.

MST experiments were performed on a Monolith NT.115 instrument (NanoTemper) in interaction buffer containing 20 mmol/L MOPS, pH 7, 1 mmol/L CaCl₂, 50 mmol/L KCl, 1 mmol/L DTT and 0.05% (v/v) Tween-20. Human cTnC was labelled with Alexa 647-NHS (Molecular Probes, Inc; ThermoFisher Scientific) according to the manufacturer’s instructions, and dye incorporation (efficiency of > 80%) was confirmed by HPLC and ESI–MS. Labelled cTnC was gel-filtered into the interaction buffer. Titration experiments were performed with a fixed concentration of 100 nmol/L of Alexa647-labelled cTnC in premium capillaries at a constant temperature of 25 °C.

MST experiments were performed on a Monolith NT.115 instrument (NanoTemper) in interaction buffer containing 20 mmol/l Mops, pH 7, 1 mmol/l MgCl₂, 50 mmol/l KCl, 1 mmol/l DTT, and 0.05% (v/v) Tween-20. For experiments with the isolated m-motif, the pH was adjusted to 6.2.
Proteins were labeled with Alexa 647-NHS (Molecular Probes, Inc; Thermo Fisher Scientific) according to the manufacturer's instructions, and dye incorporation (efficiency of >80%) was confirmed by HPLC and ESI–MS. All proteins were either gel-filtered into and/or extensively dialyzed against interaction buffer. Titration experiments were performed with a fixed concentration of 100 nmol/l of Alexa647-labeled proteins in premium capillaries.

Microscale thermophoresis experiments were performed on a Monolith NT.115 instrument (NanoTemper, Cambridge CB3 0AX, UK) in interaction buffer containing 20 mmol/liter MOPS, pH 7, 1 mmol/liter EDTA, 50 mmol/liter KCl, 1 mmol/liter DTT, and 0.05% (v/v) Tween 20. Myosin S2Δ was labeled with Alexa 647–NHS (Molecular Probes Inc., ThermoFisher Scientific, Paisley, UK) according to the manufacturer's instructions, and dye incorporation (efficiency of ∼80%) was confirmed by HPLC and ESI-MS. All proteins were either gel-filtered into and/or extensively dialyzed against interaction buffer. Titration experiments were performed with a fixed Alexa 647–myosin S2Δ concentration of 100 nmol/liter in standard treated capillaries. For experiments with isolated m-motif, the pH of the MST buffer was adjusted to 6.2, and experiments were performed in enhanced gradient standard capillaries.