Mass photometry was performed on a Refeyn TwoMP mass photometer. Protein stocks were brought to room temperature and diluted with stock buffer (500mM Na2HPO4, 50mM Tris, 10% glycerol, pH 7.5) to a concentration of 100nM. A 20 l droplet of each sample dilution was placed on top of a clean chambered-coverslip mounted on the oil objective of the instrument. Following autofocus stabilization, a one-minute movie was recorded using the Aquire-MP (Refeyn) software. A protein standard containing equimolar amounts of four proteins (conalbumin, aldolase, ferritin and thyroglobulin) with a molecular mass ranging from 75 to 669 kDa was also measured and used for calibration. Movies were converted to mass using a contrast-to-mass calibration and analyzed using the Discover-MP software (Refeyn).
Aquiremp
Manufactured by Refeyn
The AquireMP is a mass photometry instrument designed to analyze the mass and size of biomolecules. It utilizes a proprietary optical technique to provide accurate, label-free measurements of molecular mass and size distribution in solution. The AquireMP is a versatile tool that can be used to study a wide range of biomolecules, including proteins, nucleic acids, and protein complexes.
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5 protocols using aquiremp
1
Mass Photometry of Protein Samples
2
Stoichiometry of Mrp: IgG Complex
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The stoichiometry of Mrp: IgG was assessed using mass photometry. Samples containing Mrp216 and IgG at a 1:1, 1:10, and 10:1 M ratio in PBS (10ul of 100 nM Mrp216–10ul of 100 nM IgG) were prepared, and 10ul of each sample was analyzed over 10 min at a rate of 600 frames/min with an ONEMP mass photometer (Refeyn LTD). Mass photometry experiments were performed in duplicate. Data were obtained and analyzed using AquireMP and DiscoverMP, version 1.2.3 (Refeyn LTD) as previously described (57 ). M1 and M54 proteins were used as positive and negative controls, respectively.
3
Mass Photometry of Recombinant C9 Variants
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Mass photometry was conducted on a TwoMP instrument (Refeyn) operating at room temperature on an active anti-vibration platform. Briefly, calibration standards were measured on the day of the experiment in 20 mM HEPES, pH 7.2, 150 mM NaCl, followed by purified recombinant variants of C9. Stock solutions of roughly 100 nM were directly diluted to 5–10 nM and measurements were repeated for a minimum of two replicates. Mass photometry images were acquired and analysed using the Refeyn AquireMP and DiscoverMP packages (v2.5) respectively. Gaussian fit was performed in GraphPad Prism.
4
Interferometric Scattering Microscopy for MinDE Landing Assays
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Imaging of MinDE landing assays was performed on a custom-built interferometric scattering microscope described in refs. 17 (link),54 (link) with a 445-nm laser diode for illumination and 635 nm for focus stabilization. Image acquisition was controlled using custom-written software in Labview described in ref. 17 (link). Landing events were recorded at a frame rate of 1 kHz for 60 s per video. Videos were saved fivefold frame-averaged (200 Hz effective frame rate) and threefold pixel binned (70.2 nm effective pixel size).
SLB experiments were carried out on a commercial Refeyn OneMP mass photometer (Refeyn Ltd). Videos were acquired for either 45 s (landing assay of mass standards, Fig.1 ) or 350 s (SLB assays) with the AquireMP (Refeyn Ltd, v.2.3) software at a frame rate of 1 kHz. Videos were saved fivefold frame-averaged (200 Hz effective frame rate) and fourfold pixel-binned (84.4 nm effective pixel size). Measurements were performed at room temperature (23 ± 2 °C).
SLB experiments were carried out on a commercial Refeyn OneMP mass photometer (Refeyn Ltd). Videos were acquired for either 45 s (landing assay of mass standards, Fig.
5
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.
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