Purified POT1/TPP1 (5 mM) and CST (7.5 mM) proteins were mixed with 7.5 mM [GGTTAG] 3 in MP buffer (20 mM HEPES pH 7.5, 150 mM NaCl, and 0.1 mM TCEP pH 7.5, filtered 3x through 0.22 mm syringe filter) and incubated for 1 h at 4 C. Where indicated, lPP treatment of POT1/TPP1 proteins was performed as described above prior to incubation with CST. Immediately prior to measurement, samples were diluted to 100 nM POT1/TPP1 in MP buffer, then 2 mL was added to 8 mL MP buffer in a sealed well on a glass slide for a final concentration of 20 nM POT1/TPP1 and 1.5-fold excess CST and ssDNA. Data were collected using a OneMP mass photometer (Refeyn) calibrated with bovine serum albumin (66 kDa), beta amylase (224 kDa), and thyroglobulin (660 kDa). Movies were acquired for 6,000 frames (60 s) using AcquireMP software (v2022R1) and default settings. Final protein concentrations were empirically determined to achieve $50 binding events per second. Raw event measurements were converted to frequency distributions using DiscoverMP software (v2022R1) or Prism 9 (GraphPad) and a bin size of 10 kDa. Gaussian curves were autofitted with DiscoverMP to provide total peak counts for quantification.
Onemp mass photometer
Manufactured by Refeyn
Sourced in United Kingdom
The OneMP mass photometer is a compact and versatile instrument designed for accurate and reproducible measurement of the mass-to-charge ratio (m/z) of molecules in a sample. It utilizes a combination of optical and mass spectrometry techniques to provide detailed information about the molecular composition of complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Discovermp, by Refeyn (13 mentions)
Acquiremp, by Refeyn (12 mentions)
Discovermp software, by Refeyn (9 mentions)
Acquiremp software, by Refeyn (6 mentions)
Prism 9, by GraphPad (3 mentions)
Nativemark protein standard, by Thermo Fisher Scientific (3 mentions)
Nativemark unstained protein standard, by Thermo Fisher Scientific (3 mentions)
Precleaned coverslips, by Refeyn (2 mentions)
Unstained protein standard, by Thermo Fisher Scientific (2 mentions)
Culturewell reusable gaskets cw 50r 1, by Grace Bio-Labs (2 mentions)
Culturewell gaskets, by Merck Group (2 mentions)
Aquiremp, by Refeyn (2 mentions)
Culturewell gasket, by Grace Bio-Labs (1 mentions)
Microscope coverslips, by Marienfeld (1 mentions)
A8781, by Merck Group (1 mentions)
Discover mp v2, by Refeyn (1 mentions)
Microscope coverslips, by Carl Roth (1 mentions)
Gbl103250, by Grace Bio-Labs (1 mentions)
Nativemark unstained protein marker, by Thermo Fisher Scientific (1 mentions)
Nativemark, by Thermo Fisher Scientific (1 mentions)
51 protocols using onemp mass photometer
1
Quantification of POT1/TPP1-CST Complexes
2
Tau Protein Mass Analysis by OneMP
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Mi and Ms data were acquired in Refeyn OneMP mass photometer. Prior to the measurement, 50 uM tau Mi was diluted 900X times to 55.5 nM and 200 nM Ms was diluted 30X times to 6.6 nM in 1xPBS (sigma 45ZP17). 15 µl of each diluted tau sample was injected into the flow-chamber and movies of either 60 or 90 s duration were recorded after autofocus stabilization. Data was processed by Refeyn team through Gaussian fitting to provide the peak mass, the sigma (standard deviation) of the Gaussian, and the number of particles under that Gaussian (and as a % of all counts in the graph). Percentages are with respect to all the particles contained in the graph. Overlapping Gaussian curves will over-count the number of particles in a given population.
3
Mass Photometry Analysis of SLFN11
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The molecular mass of SLFN11 in solution was determined by mass photometry. All mass photometry measurements were carried out using a OneMP mass photometer (Refeyn). Prior to each measurement the focus was adjusted by applying 19 μl mass photometry buffer (25 mM Tris pH 7.5, 2 mM MgCl2, 1 mM DTT with variable concentrations of NaCl) to a new flow chamber. SLFN11 was diluted in sterile filtered mass photometry buffer to a final concentration of 50 nM, immediately prior to mass photometry measurements. For ssDNA stabilization experiment, 60 nt ssDNA was added with a final concentration of 100 nM or 300 nM. Movies were recorded for 60 s and data were analysed using AcquireMP (Refeyn) 2.3.
4
Mass Photometry Analysis of Protein Complexes
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Mass photometry was performed using OneMP mass photometer (Refeyn Ltd, Oxford, UK)
and data acquisition performed using AcquireMP (Refeyn Ltd, Oxford, UK). Microscope Values in parentheses correspond to the statistics in the highest resolution bin. RMSD, rootmean-square deviation. LD corresponds to the 'loop deletion' variant of PPM1H. R-merge = S hkl S j ½I hkl,j -½/S hkl S jhkl,j R-work = S hkl ½F o,hkl -F c,hkl ½/S hkl F o,hkl
and data acquisition performed using AcquireMP (Refeyn Ltd, Oxford, UK). Microscope Values in parentheses correspond to the statistics in the highest resolution bin. RMSD, rootmean-square deviation. LD corresponds to the 'loop deletion' variant of PPM1H. R-merge = S hkl S j ½I hkl,j -½/S hkl S jhkl,j R-work = S hkl ½F o,hkl -F c,hkl ½/S hkl F o,hkl
5
Mass Photometry Analysis of Protein Conjugates
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Protein conjugation samples for MP analysis were diluted with PBS (46-013-CM, Corning, NY) to a concentration of approximately 20 nM. The AAV conjugation sample was diluted to 10 11 particles per milliliter immediately prior to MP measurements. The current study used OneMP mass photometer and precleaned coverslips (Refeyn, UK). Following the standard protocol [23] (link), 10 μL of filtered PBS was loaded into the sample well for MP focusing, and then 10μL of the diluted sample was added and mixed with the buffer in the same well. Data were collected for one minute immediately after the mixing using the AcquireMP software (Refeyn, UK). BSA conjugation samples were measured using the standard MP detection area, while the virus conjugation samples were measured under the expanded detection area. The MP signals in both detection modes were calibrated, respectively, using BSA (05470-1G, MilliporeSigma, MA) and the Unstained Protein Standard (LC0725, ThermoFisher, MA). MP data were processed in the DiscoverMP software (Refeyn, UK).
6
Mass Photometry Characterization of Protein and Viral Conjugates
7
Mass Photometry Analysis of CODV Complexes
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All samples were subjected to analysis by mass photometry33 (link). Here, 100 nM of CODVIg, CODVIg:IL4, CODVIg:IL13, CODVIg:IL4:IL13 and CODVIg:IL13:RefAbFab were analysed using a OneMP mass photometer (Refeyn Ltd, Oxford, UK), previously calibrated with SB buffer to find the focal point and to estimate background noise. Subsequent to the measurements, image processing was performed as described in Sonn-Segev et al.33 (link) using the DiscoverMP (Refeyn Ltd) software package and graphs of intensity vs molecular weight plotted.
8
Mass Photometry Experimental Protocol
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Mass photometry experiments were performed on a Refeyn OneMP mass photometer (Refeyn) similar to described before51 (link). Microscope coverslips (24 mm × 50 mm; Marienfeld) were prepared by sequential cleaning in sonication baths of isopropanol and then MilliQ water (2x), followed by placement of a CultureWell gasket (Grace Biolabs). About 15 µL of PBS was placed in a well for focusing, after which about 3 µL of diluted sample was mixed in. Measurement concentrations were typically around 5–20 nM. Measurements were recorded using medium field-of-view settings for 120 s. An in-house calibration mix consisting of IgG4Δhinge-L368A (73 kDa), IgG1-Campath (149 kDa), apoferritin (483 kDa), and GroEL (800 kDa) was used. Recordings were processed in DiscoverMP (Refeyn), from which also the relative abundances of protein complexes were obtained. Further data analysis and plotting were performed in Jupyter Notebook using an in-house Python library.
9
Protein Stoichiometry Analysis using Refeyn OneMP
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The Refeyn OneMP mass photometer was used to determine stoichiometry of protein isolates in solution. To calibrate the instrument, Native Mark protein standard (Biorad) was diluted 50 fold in sample buffer at room temperature. 2 μl of diluted calibration mixture was mixed with 18 μl of sample buffer on silicone wells on a cleaned microscope slide (170 ± 5 μm thickness, Marienfeld). We used the 66, 146, 480 and 1048 kDa peaks for a four-point calibration. For the measuements, 18 μl buffer were pre-loaded into a silicone well, then 2 μl of 500 nM protein solution was mixed in prior to acquisition, yielding a final concentration of 50 nM. We collected 6000 frames for each protein using default instrument parameters. Data was analyzed with the DiscoverMP software provided by Refeyn, using default parameters for event extraction and fitting. Frames affected by strong vibration or aggregates moving across the image were manually excluded.
10
Mass photometry of gel filtration samples
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Mass photometry data was collected using a OneMP mass photometer (Refeyn). 15 μL of detergent-free gel filtration buffer [20 mM Tris, 150 mM NaCl, 5 mM MgCl2 pH 8.3] was applied to a coverslip, and, after focusing, 3 μL of the undiluted gel filtration fraction was added to the drop and mixed. Movies were acquired for 6,000 frames (60 seconds) using AcquireMP software with the large view setting. Raw data was processed using DiscoverMP software. For calibration, a mixture of beta amylase (Sigma cat. A8781) and thyroglobulin (Sigma cat. T9145) was used.
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