Mass photometry measurements were performed on a 2MP-0132 mass photometer (Refeyn Ltd). For the measurements, coverslips (No. 1.5 H thickness, 24 × 50 mm, VWR) were cleaned by dipping it into iso-propanol and Milli-Q water followed by drying under a stream of gaseous nitrogen. Subsequently, silicone gaskets (CultureWellTM Reusable Gasket, Grace Bio-Labs) were placed on the cleaned coverslips to create wells for samples. For mass measurements, gaskets were filled with 18 μl protein elution buffer to allow focusing the microscope onto the coverslip surface. Subsequently, 2 μl (50 nM) protein solution was added into the 18 μl droplets and mixed. A movie was recorded for 1 min using the software AcquireMP (Refeyn Ltd). Data analysis was performed using DiscoverMP (Refeyn Ltd). To convert the measured optical reflection-interference contrast into a molecular mass, a known protein size marker (NativeMarkTM Unstained Protein Standard, Invitrogen) was used.
Culturewell reusable gasket
Manufactured by Grace Bio-Labs
Sourced in United States
The CultureWell Reusable Gasket is a laboratory equipment component designed to provide a sealed, airtight connection between chambers or vessels. It is made of durable, high-quality materials and can be reused multiple times.
Automatically generated - may contain errors
Lab products found in correlation
Discovermp, by Refeyn (6 mentions)
Acquiremp, by Refeyn (5 mentions)
Nativemark unstained protein standard, by Thermo Fisher Scientific (4 mentions)
2mp 0132 mass photometer, by Refeyn (2 mentions)
Onemp, by Refeyn (1 mentions)
No 1.5h, by Marienfeld (1 mentions)
Glass coverslip, by Avantor (1 mentions)
Onemp mass photometer, by Refeyn (1 mentions)
Acquiremp software, by Refeyn (1 mentions)
Glass coverslip, by Marienfeld (1 mentions)
Twomp, by Refeyn (1 mentions)
Thyroglobulin, by Merck Group (1 mentions)
Biolever mini bl ac40ts c2, by Olympus (1 mentions)
Apdmes, by ABCR (1 mentions)
9 protocols using culturewell reusable gasket
1
Mass Photometry for Protein Characterization
2
Mass Photometry Analysis of Protein Samples
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All mass photometry measurements were executed on a Refeyn OneMP instrument. The calibration was done with a native marker protein standard mix (NativeMark Unstained Protein Standard, Thermo Scientific), which contains proteins ranging from 20 to 1,200 kDa. Coverslips (24 × 50 mm, No. 1.5H, Marienfeld) were cleaned by sequential sonication in Milli-Q water, isopropanol and Milli-Q-water, followed by drying with nitrogen. For each acquisition 2 μL of protein solution was applied to 18 μL PBS buffer, pH 7.4 in a gasket (CultureWellTM Reusable Gasket, Grace Bio-Labs) on a coverslip. Increasing working concentrations tested included 25, 50, 75 to 100 nM. Movies were recorded at 999 Hz with an exposure time of 0.95 ms by using the AcquireMP software. All mass photometry movies were processed and analysed in the DiscoverMP version 2.0 software. Samples were measured in duplicates.
3
Mass Photometry for Protein Characterization
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Mass photometry measurements were performed on a 2MP-0132 mass photometer (Refeyn Ltd). For the measurements, coverslips (No. 1.5 H thickness, 24 × 50 mm, VWR) were cleaned by sequential dipping in Milli-Q-water, isopropanol and Milli-Q-water followed by drying under a stream of gaseous nitrogen. Subsequently, silicone gaskets (CultureWell™ Reusable Gasket, Grace Bio-Labs) were placed on the cleaned coverslips to create wells for sample loading. For mass measurements, gaskets were filled with 18 μl equilibration buffer (50.mM Tris–HCl, pH 7.5 and 100 mM NaCl) to allow focusing the microscope onto the coverslip surface. Subsequently, 2 μl protein solution was added into the 18 μl droplets and mixed to obtain 50–200 nM final protein concentration. Sample binding to the coverslip surface was monitored by recording a movie for 1 minute using AcquireMP (Refeyn Ltd) software. Data analysis was performed using DiscoverMP (Refeyn Ltd). To convert the measured optical reflection-interference contrast into a molecular mass, a known protein size marker (NativeMark™ Unstained Protein Standard, Invitrogen) was measured.
4
Mass Photometry Characterization of Protein Complexes
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Mass photometry measurements were executed on a OneMP device (Refeyn Ltd.). Glass coverslips (no. 1.5 H thickness, 24 mm by 50 mm; VWR) were cleaned by 15 min of sonication in isopropanol and deionized water. Afterward, coverslips were dried under a clean stream of nitrogen. For each measurement, the cleaned coverslip was placed onto the objective. For sample delivery, a silicone gasket (CultureWell Reusable Gasket, Grace Bio-Labs) with four wells was fixed on the surface of the coverslip.
Before each measurement, all samples were incubated in measurement buffer [25 mM tris-HCl (pH 7.5), 100 mM NaCl, 3 mM EDTA, and 1 mM DTT] at 4°C for 1 hour. For data acquisition, the gasket well was filled with 9 μl of measuring buffer (room temperature) to allow surface focusing. After that, 1 μl of protein sample was added to the gasket, resulting in a final sample concentration of 5 to 15 nM (5/10 nM BLMP and 5/10 nM TOPBP1). Protein binding to the coverslip surface was monitored for 100 s using AcquireMP (version 2.3.0; Refeyn Ltd.). Data analysis was performed by DiscoverMP (version 2.3.0; Refeyn Ltd.) and OriginPro 2017. For contrast to mass conversion, a known mass standard calibrant (NativeMark Unstained Protein Standard, Invitrogen) was measured the same day. All samples were measured at least three times.
Before each measurement, all samples were incubated in measurement buffer [25 mM tris-HCl (pH 7.5), 100 mM NaCl, 3 mM EDTA, and 1 mM DTT] at 4°C for 1 hour. For data acquisition, the gasket well was filled with 9 μl of measuring buffer (room temperature) to allow surface focusing. After that, 1 μl of protein sample was added to the gasket, resulting in a final sample concentration of 5 to 15 nM (5/10 nM BLMP and 5/10 nM TOPBP1). Protein binding to the coverslip surface was monitored for 100 s using AcquireMP (version 2.3.0; Refeyn Ltd.). Data analysis was performed by DiscoverMP (version 2.3.0; Refeyn Ltd.) and OriginPro 2017. For contrast to mass conversion, a known mass standard calibrant (NativeMark Unstained Protein Standard, Invitrogen) was measured the same day. All samples were measured at least three times.
5
Mass Photometry Analysis of TnpB RNP Complex
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Measurement coverslips (no. 1.5 H, 24 × 50 mm; Marienfeld) were cleaned by sequential sonication for 5 min in MilliQ water, isopropanol and MilliQ water and then dried using a clean stream of nitrogen gas. A prepared coverslip was mounted onto the OneMP mass photometer (Refeyn) and a CultureWell Reusable Gasket (Grace Bio-Labs) was placed on top. A gasket well was filled with 10 µl of 20 mM Tris-HCl (pH 8.0 at 25 °C) and 250 mM NaCl buffer, 10 µl of the diluted TnpB RNP complex sample (approximately 60 nM) was added and the adsorption of biomolecules was monitored for 120 s using the AcquireMP software (Refeyn). For converting the measured ratiometric contrast into molecular mass, Un1Cas12f1 protein19 (link) and its oligomers ranging from 60 kDa to 250 kDa (monomer to tetramer) were used for calibration. Samples were measured in triplicates. Mass photometry movies were analysed using DiscoverMP (Refeyn).
6
Mass and Oligomerization Measurements
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Mass and oligomerization measurements were performed on glass coverslips (no. 1.5 H, 24 × 50 mm, Marienfeld) with a CultureWell reusable gasket (Grace Bio-Labs) placed on top and recorded on a mass photometer (TwoMP, Refeyn). The gasket well was filled with the sample (25 to 100 nM) and data acquisition was performed using AcquireMP (Refeyn) for 60 s. Each measurement was repeated at least three times. The recorded videos were analyzed using DiscoverMP (Refeyn) where the data were processed and fitted with Gaussian function. The molecular mass was obtained by contrast comparison with bovine serum albumin standard calibrants measured on the same day.
7
Purification and Mass Photometry of HilD Proteins
8
Mass Photometry of Ct FDO Proteins
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Data were collected on a Refeyn OneMP instrument using the DiscoverMP software (version 2.2.1). The measurements were performed using clean cover slips (High Precision cover slips, No. 1.5, 24 × 50 mm, Marienfeld) mounted with silicon gaskets (CultureWell Reusable Gaskets, Grace Biolabs). Samples were diluted in 25 mM Tris–HCl, 75 mM NaCl pH 7.5 to final concentrations of 1.7 µg ml−1 (20 nM) and 1.6 µg ml−1 (24 nM) for CtFDO and CtFDOdegl, respectively. Data were acquired and analysed using DiscoverMP (version 2.3.dev12) using default settings.
9
Covalent Immobilization of Dockerin Modules
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