Silicone gasket, by Grace Bio-Labs - Product details

1

Virus Immobilization Methods

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Unless otherwise specified, viruses were immobilized using poly-L-Lysine. Coverslips (25x65mm, thickness number 1, VWR) were heated in a furnace at 500°C for 1 hour to burn off any dust. A silicone gasket (Grace Bio-Labs, USA) was added to the glass slide and 0.01% poly-L-Lysine (Sigma) solution was added to the wells for 30 minutes. The excess was removed and the chambers were washed three times with MilliQ water and the slide allowed to dry. As an alternative chitosan was used in place of poly-L-Lysine (0.015% chitosan powder (Sigma) in 0.1M ethanoic acid).
For specific immobilisation of viruses via biotinylation, passivated microscope slides were prepared by washing in acetone and Vectabond solution (Vector Laboratories) before being incubated with NHS-PEG:Biotin-NHS-PEG in an 80:1 ratio. 0.5 mg/mL neutravidin was incubated for 10 minutes at room temperature on the slide shortly before virus was added. Viruses were biotinylated by incubation in a 1 mg/mL Sulfo-NHS-LC-Biotin (ThermoFisher) for 3 hours at 37°C before being fixed and immunolabelled as described below.

McMahon A., Andrews R., Groves D., Ghani S.V., Cordes T., Kapanidis A.N, & Robb N.C. (2023). High-throughput super-resolution analysis of influenza virus pleomorphism reveals insights into viral spatial organization. PLOS Pathogens, 19(6), e1011484.

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2

Microtubule Destabilization in Xenopus and HeLa Cells

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Xenopus S3 or HeLa cells were grown on acid-washed, poly-lysine–coated coverslips affixed with a silicone gasket (Grace Bio-Labs, Inc.). To destabilize microtubules (Fig. 5 A), cells were washed into ice-cold growth media and incubated in an ice water bath for 5 min immediately before fixation. Untreated control cells were fixed simultaneously.
100 µM monastrol treatment was performed for 1 h and fixed with 4% paraformaldehyde in PHEM at RT for 20 min. Coverslips were washed three times with TBS + 0.05% Tween and stored at 4°C until ready for use in PLA.

Banerjee B., Kestner C.A, & Stukenberg P.T. (2014). EB1 enables spindle microtubules to regulate centromeric recruitment of Aurora B. The Journal of Cell Biology, 204(6), 947-963.

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3

Surface Functionalization of Glass Coverslips

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Borosilicate glass coverslips (1.5 MenzelGläzer, Germany) were sonicated for 30 min in a 2% (V/V) solution of Hellmanex III (Helma Analytics, Germany)/deionized water. After being thoroughly rinsed with deionized water, the coverslips were dried, disposed into a plasma cleaner (Harrick Plasma, NY, USA), and exposed to a nitrogen plasma for 30 min. The coverslips are subsequently immerged into a 1% (V/V) solution of Vectabond (product code SP-1800, Vector Labs, CA, USA)/acetone for 10 min. The coverslips were then rinsed in deionized water and dried with a stream of nitrogen gas. After disposing a silicone gasket (103280, Grace Bio-Labs, OR, USA) on each coverslip, each well was filled with 20 µl of a 100 mg/ml solution of methoxy-PEG (5 kDa)-SVA/ biotin-PEG (5 kDa)-SC (2.5% (w/w) (Laysan Bio, AL, USA) in 50 mM MOPS-KOH buffer, pH 7.5 The wells were incubated for ~ 1.5 h and thoroughly rinsed with a 1 × phosphate-buffered saline (PBS; Sigma Aldrich, UK) solution. The coverslips were stored at 4 °C up to 2 weeks before use.

Dulin D., Bauer D.L., Malinen A.M., Bakermans J.J., Kaller M., Morichaud Z., Petushkov I., Depken M., Brodolin K., Kulbachinskiy A, & Kapanidis A.N. (2018). Pausing controls branching between productive and non-productive pathways during initial transcription in bacteria. Nature Communications, 9, 1478.

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4

Mass Photometry of Diluted Protein Samples

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High-precision microscope coverslips (no. 1.5, 24 × 50 mm) were cleaned with Milli-Q water, 100% isopropanol, Milli-Q water, and dried. Silicone gaskets (103250; Grace BioLabs) were placed on the coverslips. Samples were cross-linked with 0.01% glutaraldehyde for 5 min at 4°C and quenched by addition of 50 mM Tris-HCl, pH 7.5, for 1 h at 4°C. Immediately before mass photometry measurements, samples were diluted to 100 nM in buffer containing 25 mM Hepes, pH 8, 250 mM NaCl, and 2 mM DTT. For each acquisition, 20 nM of diluted protein was measured following manufacturer’s instructions. All data was acquired using a One^MP mass photometer instrument (Refeyn) and AcquireMP software (Refeyn, v2.4.1). Videos were recorded in the regular field of view using default settings. Data was analyzed using Discover^MP software (Refeyn, v2.4.2).

Abad M.A., Gupta T., Hadders M.A., Meppelink A., Wopken J.P., Blackburn E., Zou J., Gireesh A., Buzuk L., Kelly D.A., McHugh T., Rappsilber J., Lens S.M, & Jeyaprakash A.A. (2022). Mechanistic basis for Sgo1-mediated centromere localization and function of the CPC. The Journal of Cell Biology, 221(8), e202108156.

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5

Tethering Giant Unilamellar Vesicles

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For Eps15-only experiments, the GUV lipid mixture was 98 mol% DOPC, 2 mol% DOGS- NTA-Ni, and 1 mol% DP-EG10 biotin (for tethering to coverslips). For all other GUVs, the lipid mixture was or 79 mol% DOPC, 15 mol% DOPS, 1 mol% DP-EG10 biotin, and 5mol% PtdIns(4,5)P₂. Labeled GUVs also incorporated 0.1 mol% Texas Red DHPE. Lipid mixtures were spread in a film on an indium tin oxide-coated glass slide and dried under vacuum overnight. Electroformation was performed at 55°C in 350 milliosmolar glucose solution. The osmolality of the GUV solution and experimental buffer was measured using a vapor pressure osmometer (Wescor). GUVs were tethered to glass coverslips by the following process: wells were made by placing 0.8 mm thick silicone gaskets (Grace Bio-Labs) onto ultra-clean coverslips. PEG (5 kDa) was conjugated to PLL via a reaction of an amine-reactive succinimidyl valeric acid (SVA) group on PEG and primary amines in PLL. 2% of the 5 kDa PEG had a covalently attached biotin group. Wells were incubated in a 2% biotinylated PLL-PEG solution for 20 min, then washed 8x. Wells were then incubated in a 0.2 mg/ml neutravidin solution for 10 min and washed 8x. GUVs were added to wells at an 8x dilution for 10 min, then washed gently 8x.

Day K.J., Kago G., Wang L., Richter J.B., Hayden C.C., Lafer E.M, & Stachowiak J.C. (2021). Liquid-like protein interactions catalyze assembly of endocytic vesicles. Nature cell biology, 23(4), 366-376.

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6

Mass Photometry Analysis of LceB Proteins

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Mass photometry experiments for 6His‐LceB (22–366), LceB (22–366), and LceB full‐length were performed using a Refeyn One Mass Photometer machine and AcquireMP 2023.1.1 software (Refeyn Ltd). 6His‐LceB and LceB were analyzed in PBS pH 7.4 and 50 mM Tris 750 mM NaCl pH 7.5, full‐length LceB was analyzed in PBS pH 7.4. Measurements were taken in 3 mm × 1 mm silicone gaskets (Grace Bio‐Labs) and 1.5H high‐precision coverslip glass (24 × 50 mm, Deckgläser and Thorlabs). Briefly, coverslips were sonicated for 10 min in Milli‐Q water, then isopropanol, and then Milli‐Q water again. Slides were dried under air stream. The calibration curve was generated using bovine serum albumin (BSA), alcohol dehydrogenase, beta‐amylase, apoferritin and thyroglobulin in both PBS pH 7.4 and 50 mM Tris 750 mM NaCl pH 7.5. Protein samples were added to gaskets filled with the appropriate buffer and were diluted to protein concentrations ranging from 0.13 to 5 nM to optimize signal. Final concentrations used were 6His‐LceB (0.15 nM), LceB no HT (0.12 nM), and LceB full length was 5 nM. Higher concentrations of either LceB (22–366) construct saturated the detector. Images were acquired for 1–2 min depending on the sample with a 331 or 477 nm laser. DiscoverMP software 2023.1.2 (Refeyn Ltd) was used to visualize and analyze the measurements.

Penner T.V., Lorente Cobo N., Patel D.T., Patel D.H., Savchenko A., Brassinga A.K, & Prehna G. (2024). Structural characterization of the Sel1‐like repeat protein LceB from Legionella pneumophila. Protein Science : A Publication of the Protein Society, 33(3), e4889.

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7

Tethering Giant Unilamellar Vesicles

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For Eps15-only experiments, the GUV lipid mixture was 98 mol% DOPC, 2 mol% DOGS- NTA-Ni, and 1 mol% DP-EG10 biotin (for tethering to coverslips). For all other GUVs, the lipid mixture was or 79 mol% DOPC, 15 mol% DOPS, 1 mol% DP-EG10 biotin, and 5mol% PtdIns(4,5)P₂. Labeled GUVs also incorporated 0.1 mol% Texas Red DHPE. Lipid mixtures were spread in a film on an indium tin oxide-coated glass slide and dried under vacuum overnight. Electroformation was performed at 55°C in 350 milliosmolar glucose solution. The osmolality of the GUV solution and experimental buffer was measured using a vapor pressure osmometer (Wescor). GUVs were tethered to glass coverslips by the following process: wells were made by placing 0.8 mm thick silicone gaskets (Grace Bio-Labs) onto ultra-clean coverslips. PEG (5 kDa) was conjugated to PLL via a reaction of an amine-reactive succinimidyl valeric acid (SVA) group on PEG and primary amines in PLL. 2% of the 5 kDa PEG had a covalently attached biotin group. Wells were incubated in a 2% biotinylated PLL-PEG solution for 20 min, then washed 8x. Wells were then incubated in a 0.2 mg/ml neutravidin solution for 10 min and washed 8x. GUVs were added to wells at an 8x dilution for 10 min, then washed gently 8x.

Day K.J., Kago G., Wang L., Richter J.B., Hayden C.C., Lafer E.M, & Stachowiak J.C. (2021). Liquid-like protein interactions catalyze assembly of endocytic vesicles. Nature cell biology, 23(4), 366-376.

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8

PLL-PEG Passivation for FRAP Assays

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Samples were prepared in wells formed by 1.5 mm thick silicone gaskets (Grace Biolabs) on Hellmanex II (Hellma) cleaned, no.1.5 glass coverslips (VWR) passivated with poly-L-Lysine conjugated PEG5k. A second coverslip was placed on top to seal the imaging chamber to prevent evaporation during imaging. Fluorescence microscopy was performed using the Olympus SpinSR10 spinning disc confocal microscope fitted with a Hamamatsu Orca Flash 4.0V3 SCMOS Digital Camera. FRAP was performed using the Olympus FRAP unit 405 nm laser.
PLL-PEG was prepared as described previously with minor alteration (54 ). Briefly, amine-reactive mPEG-SVA was conjugated to Poly-L-Lysine (15–30 kD) at a molar ratio of 1:5 PEG to poly-L-lysine. The conjugation reaction was performed in 50 mM sodium tetraborate pH 8.5 solution and allowed to react overnight at room temperature with continued stirring. The product was buffer exchanged into PBS pH 7.4 using Zeba spin desalting columns (7K MWCO, ThermoFisher) and stored at 4°C.

Graham K., Chandrasekaran A., Wang L., Yang N., Lafer E.M., Rangamani P, & Stachowiak J.C. (2023). Liquid-like condensates mediate competition between actin branching and bundling. bioRxiv.

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9

Curvature-sensing Protein Binding Assay

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All curvature sensing experiments were carried out in buffer containing 20 mM MOPS, 150 mM sodium chloride, 0.5 mM EGTA, and 0.5 mM EDTA (pH = 7.4). Imaging wells consisted of 0.8 mm thick silicone gaskets (Grace Bio-labs) adhered to no. 1.5 glass coverslips. Wells were passivated with buffer solution containing biotinylated PEG as described previously.¹⁴ After 20 minutes of incubation, excess biotinylated PEG was rinsed from the wells. Neutravidin was added at a concentration of 0.2 mg/mL and allowed to incubate for 10 minutes. Excess neutravidin was then rinsed from the wells using the MOPS buffer. Wells were then rinsed with a MOPS buffer solution containing 50 nM sonicated vesicles and 500 nM extruded vesicles (100 nm extruded). After 10 minutes, excess vesicles were rinsed from the wells and protein was added at specified concentrations. Protein was incubated for at least 20 minutes prior to imaging. Wells were stored in sealed containers to prevent evaporation.

Zeno W.F., Snead W.T., Thatte A.S, & Stachowiak J.C. (2019). Structured and Intrinsically Disordered Domains Within Amphiphysin1 Work Together to Sense and Drive Membrane Curvature.. Soft matter, 15(43), 8706-8717.

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10

Supported Lipid Bilayer Formation

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Small Unilamellar Vesicles (SUVs) were prepared by drying down lipid films in clean glass conical tubes. The tubes were put under vacuum for 2–3 hours. Then the lipid film was resuspended in phosphate buffered saline (PBS) to create a 0.5 mM solution of liposomes. The liposomes were sonicated with a horn sonicator for 16 minutes in 4-minute intervals separated by 2 minute cooling periods in an ice bath. Ultraclean coverslips were used as the substrate for supported lipid bilayer (SLB) preparation. Silicone gaskets (Grace Biolabs) formed a well on the coverslip surface. PBS was used to initially hydrate the coverslip; then a 0.5 mM solution of SUVs was pipetted onto the coverslip, followed by a 15-minute incubation period during which the SLB formed. The SLB was washed repeatedly with experimental buffer (20 mM MOPS 200 mM NaCl pH 7.35 with TCEP as needed to prevent protein aggregation). After washing, protein solution was pipetted onto the SLB and incubated for the experiments described below.

Houser J.R., Busch D.J., Bell D., Li B., Ren P, & Stachowiak J.C. (2016). The Impact of Physiological Crowding on the Diffusivity of Membrane Bound Proteins. Soft matter, 12(7), 2127-2134.

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12 protocols using silicone gasket

Virus Immobilization Methods

Microtubule Destabilization in Xenopus and HeLa Cells

Surface Functionalization of Glass Coverslips

Mass Photometry of Diluted Protein Samples

Tethering Giant Unilamellar Vesicles

Mass Photometry Analysis of LceB Proteins

Tethering Giant Unilamellar Vesicles

PLL-PEG Passivation for FRAP Assays

Curvature-sensing Protein Binding Assay

Supported Lipid Bilayer Formation

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