Single-molecule flow cells were prepared as previously described (Chandradoss et al., 2014 (link); Filius et al., 2020 (link)). In brief, to avoid non-specific binding, quartz slides (G. Finkerbeiner Inc) were acidic piranha etched and passivated twice with polyethylene glycol (PEG). The first round of PEGylation was performed with mPEG-SVA (Laysan Bio) and PEG-biotin (Laysan Bio), followed by a second round of PEGylation with MS(PEG)4 (ThermoFisher). After assembly of a microfluidic chamber, the slides were incubated with 20 μL of 0.1 mg/mL streptavidin (Thermofisher) for 2 minutes. Excess streptavidin was removed with 100 μL T50 (50mM Tris-HCl, pH 8.0, 50 mM NaCl). Next, 50 μL of 75 pM DNA-labeled peptide was added to the microfluidic chamber. After 2 minutes of incubation, unbound peptide and excess Azide-DNA from the earlier click reaction was washed away with 200 μL T50. Then, 50 μL of 10 nM donor labeled imager strands and 100 nM acceptor labeled imager strands in imaging buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.8% glucose, 0.5 mg/mL glucose oxidase (Sigma), 85 ug/mL catalase (Merck) and 1 mM Trolox (Sigma)) was injected. All single-molecule FRET experiments were performed at room temperature (23 ± 2°C).
Biotin peg
Manufactured by Laysan Bio
Biotin-PEG is a chemical compound consisting of a biotin molecule covalently linked to a polyethylene glycol (PEG) polymer. It is commonly used as a versatile linker in various biotechnological and biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin, by Thermo Fisher Scientific (5 mentions)
Trolox, by Merck Group (5 mentions)
Glucose oxidase, by Merck Group (4 mentions)
Catalase, by Merck Group (4 mentions)
Ms peg 4, by Thermo Fisher Scientific (3 mentions)
Mpeg sva, by Laysan Bio (3 mentions)
Coverslips, by Marienfeld (2 mentions)
Phosphocreatine, by Merck Group (1 mentions)
Cyclooctatetraene, by Merck Group (1 mentions)
Creatine kinase, by Merck Group (1 mentions)
Digitonin, by Merck Group (1 mentions)
Ix83 celltirf, by Olympus (1 mentions)
4 nitrobenzyl alcohol, by Merck Group (1 mentions)
Protocatechuate 3 4 dioxygenase, by Merck Group (1 mentions)
Flash 4.0 v3, by Hamamatsu Photonics (1 mentions)
Protocatechuic acid, by Merck Group (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Sulfuric acid, by Thermo Fisher Scientific (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Ultra 897, by Oxford Instruments (1 mentions)
8 protocols using biotin peg
1
Preparation of Single-Molecule Flow Cells
2
Single-Molecule FRET Microscopy Sample Preparation
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Single-molecule flow cells were prepared as previously described (Chandradoss et al., 2014; (link)Filius et al., 2020) (link). In brief, to avoid non-specific binding, quartz slides (G. Finkerbeiner Inc) were acidic piranha etched and passivated twice with polyethylene glycol (PEG). The first round of PEGylation was performed with mPEG-SVA (Laysan Bio) and PEG-biotin (Laysan Bio), followed by a second round of PEGylation with MS(PEG) 4 (ThermoFisher). After assembly of a microfluidic chamber, the slides were incubated with 20 mL of 0.1 mg/mL streptavidin (Thermofisher) for 2 minutes. Excess streptavidin was removed with 100 mL T50 (50mM Tris-HCl, pH 8.0, 50 mM NaCl). Next, 50 mL of 75 pM DNA-labeled peptide was added to the microfluidic chamber. After 2 minutes of incubation, unbound peptide and excess Azide-DNA from the earlier click reaction was washed away with 200 mL T50. Then, 50 mL of 10 nM donor labeled imager strands and 100 nM acceptor labeled imager strands in imaging buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.8% glucose, 0.5 mg/mL glucose oxidase (Sigma), 85 ug/mL catalase (Merck) and 1 mM Trolox (Sigma)) was injected. All single-molecule FRET experiments were performed at room temperature (23 G 2 C).
3
Preparation of Single-Molecule Flow Cells
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Single-molecule flow cells were prepared as previously described. 20, (link)21 (link) In brief, to avoid nonspecific binding, quartz slides (G. Finkerbeiner Inc) were acidic piranha etched and passivated twice with polyethylene glycol (PEG). The first round PEGylation was performed with mPEG-SVA (Laysan Bio) and PEG-biotin (Laysan Bio), followed by a second round of PEGylation with MS(PEG)4 (ThermoFisher). After assembly of a microfluidic chamber, the slides were incubated with 20 µL of 0.1 mg/mL streptavidin (Thermofisher) for 2 minutes.
Excess streptavidin was removed with 100 µL T50 (50mM Tris-HCl, pH 8.0, 50 mM NaCl).
Next, 50 µL of 75 pM Cy5 labeled ssDNA was added to the microfluidic chamber. After 2 minutes of incubation, unbound ssDNA was washed away with 100 µL T50. For experiments in Figure 1, 50 µL of 10 nM donor labeled imager strands in imaging buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 100 mM MgCl2, 0.8 % glucose, 0.5 mg/mL glucose oxidase (Sigma), 85 ug/mL catalase (Merck) and 1 mM Trolox (Sigma)) was injected. All singlemolecule FRET experiments were performed at room temperature (23 ± 2 °C).
Excess streptavidin was removed with 100 µL T50 (50mM Tris-HCl, pH 8.0, 50 mM NaCl).
Next, 50 µL of 75 pM Cy5 labeled ssDNA was added to the microfluidic chamber. After 2 minutes of incubation, unbound ssDNA was washed away with 100 µL T50. For experiments in Figure 1, 50 µL of 10 nM donor labeled imager strands in imaging buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 100 mM MgCl2, 0.8 % glucose, 0.5 mg/mL glucose oxidase (Sigma), 85 ug/mL catalase (Merck) and 1 mM Trolox (Sigma)) was injected. All singlemolecule FRET experiments were performed at room temperature (23 ± 2 °C).
4
Functionalized Flow Chambers for Microscopy
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Flow chambers for microscopy were prepared as described before (7 (link),18 (link),19 (link)). Briefly, cover slips (24 × 24 mm, Marienfeld) were functionalised with biotin-PEG (Laysan Bio). A polydimethylsiloxane (PDMS) block was made using soft-lithography methods and placed on top of the cover slips, creating a 1-mm wide and 0.1-mm high channel with a volume of 1 μl. Two stretches of polyethylene tubing (PE-60: 0.76-mm inlet diameter and 1.22-mm outer diameter, Walker Scientific) were inserted into the PDMS block at the entrance and exit of the channel to allow for buffer flow. Before the start of experiments, the flow channel was incubated with blocking buffer (50 mM Tris–HCl pH 7.6, 50 mM Potassium Chloride, 2% (v/v) Tween-20) to minimise nonspecific binding of DNA or proteins to the cover-slip surface. A syringe pump (Adelab Scientific) was used to introduce solutions to the flow cell.
5
Single-Molecule Imaging of Multidrug Resistance Protein
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Single-molecule experiments were performed at room temperature (23 ± 1°C) on an objective-type total-internal-reflection fluorescence microscope (Olympus IX83 cellTIRF). Microfluidic imaging chambers were passivated with a mixture of PEG and biotin-PEG (Laysan Bio), and incubated with 0.8 µM streptavidin (Invitrogen) followed by 2 nM fluorescently labeled, His-tagged bMRP1 that had been preincubated with biotinylated anti-His6 antibodies (Invitrogen) for 1 hr on ice in a buffer containing 50 mM Tris-HCl pH 8.0, 150 mM KCl, 2 mM MgCl2, 0.06% digitonin, 0.5 mg/mL BSA, 10 mM phosphocreatine (Sigma), and 0.1 mg/mL creatine kinase (Sigma). A triplet-state quenching cocktail (Dave et al., 2009 (link)) of 1 mM cyclooctatetraene (Sigma), 1 mM 4-nitrobenzyl alcohol (Sigma), and 1 mM Trolox (Sigma), as well as an oxygen scavenging system (Aitken et al., 2008 (link)) containing 10 nM protocatechuate-3,4-dioxygenase (Sigma) and 2.5 mM protocatechuic acid (Sigma) were supplemented to the imaging buffer. ATP and/or LTC4 were included in the imaging buffer at concentrations specified in the text. Fluorescence signals were split with a W-View Gemini-2C (Hamamatsu), directed to two CMOS cameras (Flash 4.0 v3, Hamamatsu), and acquired by MetaMorph software (Molecular Devices) at a frame rate specified in the text.
6
Passivation of Silica Substrates for TIRF Microscopy
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Silica microscope slides used for TIRF experiments were prepared as described before57 (link). Briefly, fused silica slides (Plano) were cleaned in peroxomosulfuric acid (70% (v/v) sulfuric acid; Fisher Scientific, 10% (v/v) hydrogen peroxide; Sigma-Aldrich) for 30 min and washed with Millipore water under sonication. Afterwards, the slides were incubated in methanol for 20 min and sonicated for 5 min. For silane passivation, the slides were incubated in a freshly prepared N-[3-(Trimethoxysilyl)-propyl]ethyldiamine (Sigma-Aldrich) solution (2% (v/v) in methanol with 4% (v/v) acetic acid) for 20 min, rinsed with methanol five times and an additional 20 times with Millipore water. The slides were dried for 1 h at 37 °C. For PEG passivation, 100 µL of freshly prepared passivation solution (200 mg/mL methoxy-PEG succinimidyl valerate 5000 (Laysan Bio), 5 mg/mL biotin-PEG (Laysan Bio) in 1 mM NaHCO3) was sandwiched between a slide and a coverslip, incubated for 2 h and rinsed with Millipore water 20 times. The slides and coverslips were fully dried at 37 °C, vacuum sealed in plastic tubes and stored at −20 °C.
7
Single-molecule analysis of CRISPR-Cas10 RNA binding
8
Preparation of Biotin-PEG Functionalized Flow Chambers
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Flow chambers for microscopy were prepared as described before [7] , [21] (link), [43] (link). Briefly, cover slips (24 × 24 mm, Marienfeld) were functionalised with biotin-PEG (Laysan Bio). A polydimethylsiloxane (PDMS) block was made using soft-lithography methods and placed on top of the cover slips, creating a 1-mm wide and 0.1-mm high channel with a volume of 1 μL. Two stretches of polyethylene tubing (PE-60: 0.76-mm inlet diameter and 1.22-mm outer diameter, Walker Scientific) were inserted into the PDMS block at the entrance and exit of the channel to allow for buffer flow. Before the start of experiments, the flow channel was incubated with blocking buffer (50 mM Tris-HCl pH 7.6, 50 mM Potassium Chloride, 2% (v/v) Tween-20) to minimise nonspecific binding of DNA or proteins to the cover-slip surface. A syringe pump (Adelab Scientific) was used to introduce solutions to the flow cell.
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