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Twomp

Manufactured by Refeyn
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The TwoMP is a mass photometer developed by Refeyn. It is a laboratory instrument used for the analysis of molecular weight distributions of macromolecules in solution.

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Lab products found in correlation

Discovermp, by Refeyn (8 mentions) Discovermp software, by Refeyn (3 mentions) Acquiremp, by Refeyn (3 mentions) Thyroglobulin, by Merck Group (2 mentions) Acquiremp software, by Refeyn (2 mentions) Culturewell gasket, by Grace Bio-Labs (2 mentions) Hplc grade isopropanol, by Merck Group (2 mentions) Anotop 10, by Cytiva (2 mentions) Discovermp program, by Refeyn (2 mentions) High precision glass coverslips, by Marienfeld (1 mentions) Histalon gravity column purification kit, by Takara Bio (1 mentions) Amicon ultra, by Merck Group (1 mentions) No 1.5h, by Marienfeld (1 mentions) Microscope slide, by Marienfeld (1 mentions) Aquiremp, by Refeyn (1 mentions) Discovermp 2, by Refeyn (1 mentions) Culturewell reusable gasket, by Grace Bio-Labs (1 mentions) Glass coverslip, by Marienfeld (1 mentions) Urease, by Merck Group (1 mentions) Milli q water, by Merck Group (1 mentions)

24 protocols using twomp

1

Oligomerization Analysis of PI31 Proteins

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Purified His-PI31 (wildtype or V6R mutant) proteins were dialyzed against 20 mM Tris HCl (pH 7.6 at 5 °C), 20 mM NaCl for analysis of oligomerization by mass photometry (TwoMP, Refeyn). Protein concentrations were determined by BCA assay (Pierce) and converted to molar concentrations assuming 31 kDa molecular weights. PI31 was then diluted in 0.2 μm filtered PBS into a total monomer concentration of 400 nM. Cover slips were cleaned with isopropanol and MilliQ water and fitted with silicon gaskets. The mass photometer optics were focused on 16.2 μl buffer prior to the addition and mixture of 1.8 μl sample. Sample mixture into the prefocused droplet resulted in an additional one-tenth dilution (40 nM final), and recordings were initiated immediately after mixing. Bovine serum albumin and thyroglobulin oligomers were used as molecular weight standards to calibrate ratiometric contrast measurements to molecular weight. Automatic Gaussian curve fitting for peaks was performed by DiscoverMP software (Refeyn). which identified peaks centered at 36 to 37 and 56 to 59 kDa for monomers and dimers, respectively.
Hsu H.C., Wang J., Kjellgren A., Li H, & DeMartino G.N. (2023). Ηigh-resolution structure of mammalian PI31–20S proteasome complex reveals mechanism of proteasome inhibition. The Journal of Biological Chemistry, 299(7), 104862.
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2

Mass Photometry Analysis of Protein Complexes

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Mass photometry experiments were carried out using a Refeyn TwoMP (Refeyn Ltd., Oxford, UK) MP system. AcquireMP and DiscoverMP software packages were used to record movies and analyze data, respectively, using standard settings. Microscope coverslips (high precision glass coverslips, Marienfeld) were cleaned following the Refeyn Ltd. individual rinsing procedure. Reusable self-adhesive silicone culture wells (Grace Bio-Labs reusable CultureWell gaskets) were used to keep the sample droplet shape. Contrast-to-mass calibration was carried out using BSA (Albumin, Bovine Serum Fraction V, Low Heavy Metals; Millipore), giving molecular weights of 66, 132, 198, and 264 kDa. Immediately before the measurements, protein stocks were diluted directly in a buffer containing 25 mM Hepes-NaOH (pH 7.5), 125 mM NaCl, and 2 mM MgCl2. To this end, 1 to 2 μl of protein solution was added into 18 to 19 μl of analysis buffer to reach a final drop volume of 20 μl.
Ni D., Lu X., Stahlberg H, & Ekundayo B. (2023). Activation mechanism of a short argonaute-TIR prokaryotic immune system. Science Advances, 9(29), eadh9002.
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3

His-tagged NADK2 Protein Purification and Mass Photometry

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His-tagged NADK2 (aa 61–442) or its variants in pET15b plasmid were transformed in BL21 (DE3) competent E. coli cells according to the manufacturer’s instructions. Protein expression was induced with 0.5 mM IPTG, and 500 mL of bacterial cultures were grown for 48 h at 18 °C. Protein purification was performed with a HisTALON™ Gravity Column Purification Kit (TakaraBio, 635654) according to the manufacturer’s instructions. NADK2 or its variants were eluted with 8 mL of the kit’s elution buffer that contains 150 mM imidazole. Different fractions of purified protein were concentrated using Amicon Ultra (EMD Millipore, UFC501024) - 0.5 mL filters. The protein purity was determined by western blotting. NADK2 protein diluted in water at final concentration of 0.1 μM was used for analysis for Mass Photometry (Refeyn TwoMP).
For each experiment microscope coverslips were washed three times with Milli-Q water (followed by two isopropanol washes. The coverslip was then dried with nitrogen stream to remove any liquid. Using a razor blade, gasket wells were cut and put it on the clean coverslip. The protein of interest was pre-prepared as 1 μM stock in PBS and diluted 1:10 in PBS (final 0.1 μM), just before starting the measurements. 2 μL of protein was used for each measurement and movies of 60 seconds were recorded. Data were analyzed using DiscoverMP (Refeyn).
Grider A., Bailey L.B, & Cousins R.J. (1990). Erythrocyte metallothionein as an index of zinc status in humans.. Proceedings of the National Academy of Sciences of the United States of America, 87(4), 1259-1262.
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4

Mass Photometry Analysis of RAD51 Oligomerization

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Recombinant human RAD51 protein was purified as previously described [87 (link)]. Mass photometry (MP) experiments were performed using the Refeyn TwoMP mass photometry instrument (Refeyn Ltd. Oxford, UK) in buffer containing 20 mM Tris pH 7.5, 150 mM KCl, 10 mM MgCl2, 1 mM ATP, and in the presence or absence of 5 mM CaCl2. Cover slides were cleaned by sequential washing with miliQ water and 100% isopropanol twice, then with miliQ water, and subsequently dried under an air stream. Silicon buffer gaskets were rinsed sequentially with miliQ water, isopropanol, and miliQ water and dried at room temperature. Dried silicon gaskets were applied to the glass slide with mild pressure and mounted on the mass photometer. Two protein oligomer solutions, β-amylase (56, 112, and 224 kDa) and Thyroglobulin (670 kDa), were used for molecular weight calibrations. In each experiment, 400 nM of RAD51 was incubated with 20 μM of respective inhibitor for 45 or 90 min at room temperature. The protein-inhibitor solution was then diluted 4 times into the buffer-filled gasket yielding a final concentration of 100 nM RAD51 and 5 μM inhibitor. Individual molecular weights collected from 3000 frames (59.9 s) were binned in 5 kDa bins, plotted as frequency histograms, and fitted to multiple Gaussians in GraphPad Prism.
Hong L., Braden D.C., Zhao Y., Skoko J.J., Chang F., Woodcock S.R., Uvalle C., Casey A., Wood K., Salvatore S.R., Asan A., Harkness T., Fagunloye A., Razzaghi M., Straub A., Spies M., Brown D.D., Lee A.V., Schopfer F., Freeman B.A, & Neumann C.A. (2023). Small molecule nitroalkenes inhibit RAD51-mediated homologous recombination and amplify triple-negative breast cancer cell killing by DNA-directed therapies. Redox Biology, 66, 102856.
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5

Mass Photometry Sample Preparation

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MP measurements were performed with a TWOMP (Refeyn Ltd, Oxford, UK) at room temperature (18 °C). Microscope slides (24 × 50 mm, 170 ± 5 µm, No. 1.5H, Paul Marienfeld GmbH & Co. KG, Germany) were cleaned with milli-Q water, isopropanol, milli-Q water and dried with a clean nitrogen stream. Six-well reusable silicone gaskets (CultureWellTM, 50–3 mm DIA x 1 mm Depth, 3–10 µL, Grace Bio-Labs, Inc., Oregon, USA) were carefully cut and assembled on the cover slide center. After being placed in the mass photometer and before each acquisition, an 18 µL droplet of PBS was put in a well to enable focusing on the glass surface.
Gizardin-Fredon H., Santo P.E., Chagot M.E., Charpentier B., Bandeiras T.M., Manival X., Hernandez-Alba O, & Cianférani S. (2024). Denaturing mass photometry for rapid optimization of chemical protein-protein cross-linking reactions. Nature Communications, 15, 3516.
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6

Time-Resolved Mass Photometry of ParDE1

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ParDE1 expressed and purified as above provided the starting material (theoretical heterotetramer) for mass photometry experiments. A 5 ml ParDE1 sample at 5.2 mg/ml was incubated at 37°C with shaking at 180 rpm. At each time point, a 200 μl sample was snap frozen in liquid N2. Solution-phase mass determination of the ParDE1 species present in each sample was then performed using the TwoMP (Refeyn) mass photometer. Samples were diluted 1000-fold in A500, and experimental data were obtained in the form of mass photometry videos recorded for one minute using the AcquireMP v2.5 software (Refeyn) on precleaned, high sensitivity microscope slides. A mass calibration was done using bovine serum albumin, IgG, and thyroglobulin. The experimental data were then fit to this calibration, and graphs were generated using the DiscoverMP v2.5 software (Refeyn).
Beck I.N., Arrowsmith T.J., Grobbelaar M.J., Bromley E.H., Marles-Wright J, & Blower T.R. (2023). Toxin release by conditional remodelling of ParDE1 from Mycobacterium tuberculosis leads to gyrase inhibition. Nucleic Acids Research, 52(4), 1909-1929.
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7

Mass Photometry Analysis of Meprin Variants

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Mass photometry was conducted on an active anti-vibration platform using a TwoMP instrument (Refeyn; 20 °C). Meprin variants were measured at a final concentration of roughly 2–5 nM and calibration standards were measured on the day of the experiment. Mass photometry images were acquired and analysed using the Refeyn AquireMP and DiscoverMP packages (v2.5) respectively.
Bayly-Jones C., Lupton C.J., Fritz C., Venugopal H., Ramsbeck D., Wermann M., Jäger C., de Marco A., Schilling S., Schlenzig D, & Whisstock J.C. (2022). Helical ultrastructure of the metalloprotease meprin α in complex with a small molecule inhibitor. Nature Communications, 13, 6178.
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8

Mass Photometry of Ten-m Protein

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Mass photometry experiments were performed in the Biophysics core facility on a Refeyn TwoMP at the University of Illinois‐Chicago. The instrument was calibrated using β‐Amylase and thyroglobulin. First, Ten‐m samples were purified as described above, and samples were diluted in SEC buffer to various concentrations from 10 nM to 500 nM. 3,000 frame movies were collected and individual counts were recorded, and masses were extrapolated using calibration curves. The data were examined and plotted using Refeyn DiscoverMP software.
Li J., Bandekar S.J, & Araç D. (2023). The structure of fly Teneurin‐m reveals an asymmetric self‐assembly that allows expansion into zippers. EMBO Reports, 24(6), e56728.
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9

Mass Photometry of Protein Complexes

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Mass photometry experiments were performed using the Refeyn TwoMP instrument. To improve particle adsorption, coverslips were placed in a coplin jar, immersed in 1 M KOH, and placed in an ultrasonic water bath for 60 min. To generate a standard mass curve, thyroglobulin and beta-amylase were diluted to 3 nM and 10 nM, respectively, in SEC buffer. Thyroglobulin, beta-amylase dimer, and beta-amylase tetramer peaks were selected as calibrants. Lig4-XRCC4 complexes were diluted to 10 nM in SEC buffer and measurements were collected for 20 sec.
Stinson B.M., Carney S.M., Walter J.C, & Loparo J.J. (2024). Structural role for DNA Ligase IV in promoting the fidelity of non-homologous end joining. Nature Communications, 15, 1250.
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10

Mass Photometry Analysis of UBR5 Oligomeric State

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To determine the oligomeric state of UBR5, mass photometry measurements were performed on the Refeyn TwoMP using Refeyn AcquireMP 2.3.0 software. Mass calibration was achieved by measurement of a protein mixture, providing a range of molecular masses as follows: conalbumin (75 kDa), aldolase (158 kDa), ferritin (440 kDa) and thyroglobulin (669 kDa) in a final concentration of ~50 nM of each component. Measurements of either UBR5 or UBR5Dimer were carried out by diluting UBR5 to a final concentration of ~140 nM in the same buffer used for focus-finding (25 mM HEPES (pH 7.5), 150 mM NaCl and 1 mM DTT). Videos were collected for 1 min. Data were then analyzed using DiscoverMP 2.3.0 (Refeyn) software and the collected mass calibration as a reference.
Hehl L.A., Horn-Ghetko D., Prabu J.R., Vollrath R., Vu D.T., Pérez Berrocal D.A., Mulder M.P., van der Heden van Noort G.J, & Schulman B.A. (2023). Structural snapshots along K48-linked ubiquitin chain formation by the HECT E3 UBR5. Nature Chemical Biology, 20(2), 190-200.
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