Quartz coverslips were treated with 100% ethanol and 1 mM KOH. Then, aminosilanization of coverslips was carried out in a 1% (v/v) (3-Aminopropyl)triethoxy silane (Alfa Aesar, A10668, UK) solution in acetone. PEGylation was carried out by incubating mixture of biotinPEG-SVA and mPEG-SVA (Laysan Bio, AL) in the ratio of 1:10 prepared in 0.1 M NaHCO3 solution on the top of silanized coverslip for 3–4 hr. Finally, PEGylated coverslips were stored under dry nitrogen gas at −20°C.
Biotin peg sva
Manufactured by Laysan Bio
Sourced in United States
Biotin-PEG-SVA is a bifunctional crosslinking reagent that contains biotin and an N-hydroxysuccinimide (NHS) ester. The biotin moiety can bind to streptavidin or avidin, while the NHS ester can react with primary amines on proteins or other biomolecules, forming a covalent bond. This product can be used to attach biotin to proteins or other molecules for various biotechnology applications.
Automatically generated - may contain errors
Lab products found in correlation
Mpeg sva, by Laysan Bio (13 mentions)
Glucose oxidase, by Merck Group (5 mentions)
Trolox, by Merck Group (5 mentions)
Catalase, by Roche (4 mentions)
D glucose, by Merck Group (4 mentions)
Bovine serum albumin (bsa), by New England Biolabs (3 mentions)
Neutravidin, by Thermo Fisher Scientific (3 mentions)
3 aminopropyltriethoxysilane, by Thermo Fisher Scientific (2 mentions)
Anti digoxigenin coated polystyrene beads, by Polysciences (2 mentions)
Aptes, by Merck Group (2 mentions)
Peg succinimidyl valerate sva, by Laysan Bio (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Hanks balanced salt solution (hbss), by Merck Group (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Milli q water, by Thermo Fisher Scientific (1 mentions)
Methanol, by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Tris hydrochloride, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions)
18 protocols using biotin peg sva
1
Silanization and PEGylation of Coverslips
2
Biotin-Mediated Treg-Islet Co-Incubation
3
Imaging Aptamer-Bead Interactions by TIRF
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The quartz slides and coverslips, passivated with PEG (a mixture of mPEG-SVA and biotin-PEG-SVA, Laysan Bio) were assembled to form imaging chambers38 (link). For TIRF experiments, 30 pM RNA aptamer construct(s) were immobilized on the surface via biotin–neutravidin interaction. Finally, imaging buffer was added for data acquisition. Unless otherwise mentioned, the imaging buffer comprised of 50 mM Tris-HCl pH 7.5, 0.8% w/v D-glucose [Sigma], 165 U mL−1 glucose oxidase [Sigma], 2170 U mL−1 catalase [Roche], 3 mM Trolox [Sigma], and predetermined amount of KCl/MgCl2.
For integrated fluorescence-force measurements, the imaging chamber was incubated in blocking buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mg mL−1 BSA [NEB], 1 mg mL−1 tRNA [Ambion]) for 1 h. The aptamer constructs were then diluted to 10 pM and immobilized on the surface via biotin–neutravidin interaction. Subsequently, 1 μM anti-digoxigenin coated polystyrene beads (Polysciences), diluted in a buffer containing 10 mM Tris-HCl pH 7.5 and 50 mM NaCl, were added to the imaging chamber and incubated for 30 min. Finally, data were acquired in the imaging buffer.
For integrated fluorescence-force measurements, the imaging chamber was incubated in blocking buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mg mL−1 BSA [NEB], 1 mg mL−1 tRNA [Ambion]) for 1 h. The aptamer constructs were then diluted to 10 pM and immobilized on the surface via biotin–neutravidin interaction. Subsequently, 1 μM anti-digoxigenin coated polystyrene beads (Polysciences), diluted in a buffer containing 10 mM Tris-HCl pH 7.5 and 50 mM NaCl, were added to the imaging chamber and incubated for 30 min. Finally, data were acquired in the imaging buffer.
4
Biofunctionalization of Quartz Slides
5
Immobilization of Nucleosomes for Single-Molecule Studies
6
Lipid Bilayer Formation Protocol
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MOPS, Hepes, Tris hydrochloride, NaCl, DTT, IPTG, β-mercaptoethanol, thrombin protease, and Triton X-100 were purchased from Thermo Fisher Scientific. EDTA, EGTA, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), PMSF, EDTA-free protease inhibitor tablets, Thrombin CleanCleave Kit, poly-l -lysine (PLL), Atto 488 NHS-ester, and Atto 594 NHS-ester were purchased from Sigma-Aldrich. Human rhinovirus-3C (HRV-3C) protease, neutravidin, Oregon Green 488–DHPE, and Texas Red–DHPE were purchased from Thermo Fisher Scientific. mPEG–succinimidyl valerate (SVA), biotin-PEG-SVA, mPEG-silane, and biotin-PEG-silane (all PEGs were molecular weight 5,000 D) were purchased from Laysan Bio. Dipalmitoyl-decaethylene glycol-biotin (DP-EG10-biotin) was provided by D. Sasaki of Sandia National Laboratories, Livermore, CA (Momin et al., 2015 (link)). All other lipids were purchased from Avanti Polar Lipids, including l -α-phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2; from porcine brain), DOGS-NTA-Ni, DOPC, and 1,2-dioleoyl-sn-glycero-3-phospho-l -serine (DOPS; sodium salt). The lipid compositions for all experiments are listed in the figure captions.
7
Multiplex Immunoassay Bioconjugation Protocol
8
Functionalized Coverslips for Biomolecular Imaging
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Coverslips were soaked in piranha solution (25% H2O2 and 75% concentrated H2SO4) and sonicated for 1 h, followed by multiple rinses in water (Thermo Fisher Scientific, molecular-biology grade) and acetone (Thermo Fisher Scientific, HPLC grade). Dry and clean coverslips were then treated with Vectabond/acetone (1% v/v) (Vector Labs) solution for 5 min and then rinsed with water and left in a dried state until used. In order to prevent non-specific adsorption of biomolecules onto the glass surface, coverslips were functionalized prior to use with a mixture of poly(ethylene glycol) succinimidyl valerate, MW 5000 (mPEG-SVA) and biotin-PEG-SVA at a ratio of 99:1 (w/w) (Laysan Bio) in 0.1 M sodium bicarbonate (Thermo Fisher Scientific) for 3 h18 (link). Excess PEG was rinsed with water, and the coverslips were dried under a N2 stream. Imaging chambers (~5 μL) were constructed by pressing a polycarbonate film (Grace Bio-Labs) with an adhesive gasket onto a PEG-coated coverslip. Two silicone connectors glued onto the pre-drilled holes of the film served as inlet and outlet ports. The surface was incubated with 7 μL of a 2 mg/ml neutravidin solution (Thermo Fisher Scientific). Excess neutravidin was then washed off with 100 μL of 1X PBS buffer.
9
Functionalized Flow Cell Assembly
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Flow cells were prepared essentially as described previously24 . Briefly, glass coverslips (24 × 40 mm, 0.17 ± 0.01 mm, VWR, 630–2,746) were cleaned with two rounds of sonication in ethanol and 1 M potassium hydroxide, 30 min each round, treated with (3-Aminopropyl)triethoxysilane in acetone and subsequently functionalized with a mixture of polyethylene glycol (PEG)-Succinimidyl Valerate (SVA) (MW 5,000, Laysan Bio Inc.) and Biotin-PEG-SVA (MW 5,000, Laysan Bio Inc.) in 100 mM sodium bicarbonate buffer (pH 8.2). A flow channel was prepared by sandwiching double-sided tape between the functionalized coverslip and a glass slide (VWR, 48,300–025) containing two holes. An inlet (Intramedic, PE20; inner diameter 0.015″, outer diameter 0.048″) and an outlet (Intramedic, PE20; inner diameter 0.015″, outer diameter 0.048″) tubing are attached to the holes on the glass slide and sealed with epoxy (Devcon, 5 min Epoxy). The inlet tubing was submerged into an Eppendorf tube filled with buffer, while the outlet tubing was attached to an automated syringe pump (Harvard Apparatus, Pump 11 Plus Single Syringe) to withdraw buffer through the flow channel.
10
Fabricating Drift-Corrected Flow Cells
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Flow cells for use in the MT apparatus were made by sandwiching double-sided tape between two microscope coverslips. The floor surface of the flow cell was cleaned by sonication in solutions of ethanol (95%) and 1 M potassium hydroxide. Silica microbeads (3 μm diameter; Bangs Laboratories, Fishers, IN, USA) were partially melted onto the surface to create reference beads, which could be tracked to monitor and correct stage drift. The glass was then treated with silane (2% v/v solution of 3-aminopropyltriethoxysilane in acetone), which was then cured at 110°C for 1 h. This coverslip was then functionalized with a layer of biotinylated polyethylene glycol (MPEG-SVA and Biotin-PEG-SVA; mol wt = 5000; Laysan Bio, Arab, AL, USA). This functionalization allowed us to attach DNA tethers to the floor of the chamber via biotin-streptavidin bonds.
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