Polylink protein coupling kit

Carboxylated microparticles (Polybead® Carboxylate Microspheres 3 μm; Polysciences, Inc, Warrington, PA) were functionalized with an Alexa Fluor® 488 conjugated goat anti-Rabbit IgG H&L antibody (Life Technologies, Carlsbad, CA) using the PolyLink Protein Coupling kit (Polysciences, Inc.), following the manufacturer’s instructions (Alexa488-IgG-microparticles, from now on).

To estimate the force-extension curve of the nanospring, biotin-modified staples and digoxigenin (DIG)-modified staples (Supplementary Data) were added to the 100 nM core staples, which were then folded to attach biotin and DIG at opposite ends of the nanospring.
Carboxylate-modified polystyrene beads (0.2 μm in diameter, Invitrogen) were crosslinked to anti-DIG polyclonal antibody (Roche) and BSA using the Polylink Protein Coupling Kit (Polysciences, Inc.). One microlitre of anti-DIG antibody-coated beads (∼3 nM) and 10 μl of nanospring (∼1 nM) were mixed and incubated for 1 h on ice before use in an optical tweezers assay.
A single flow chamber was made using double-sided transparent tape (Scotch) and coverslips (Matsunami). Five microlitres of neutravidin (5 mg ml⁻¹, Invitrogen) was flowed into a chamber and incubated for 3 min. Unbound neutravidin was washed out by Assay buffer (AB; 20 mM HEPES-KOH pH 7.8, 25 mM KCl, 5 mM MgCl₂ and 1 mM EGTA) and 10 μl of biotin-coated bead (∼3 pM and 0.2 μm in diameter, Invitrogen) was flowed into the cell, which was then incubated for 5 min. Twenty microlitres of bead-nanospring (bead-NS) was diluted ten times in AB plus an oxygen scavenger system44 (link) and flowed into the cell, which was then sealed with nail polish. The chamber was observed by our microscope after 5 min.

For the generation of P-selectin-coupled beads polybead-carboxylate-microspheres (2 µm, 2.7% solids (w/v) Polysciences), polylink protein coupling kit (Polysciences), and recombinant human P-selectin protein (carrier free, Catalog no. ADP3-050, R&D Systems) were used. Coupling was performed according to the manufacturer’s protocol. About 3.1 mg microparticles were centrifuged (1000g, 10 minutes) resuspended in polylink coupling buffer twice before 21-mg carbodiimide was added for activation. After 15 minutes, 50-µg P-selectin was added and incubated for 120 minutes. Then, beads were centrifuged (1000g, 10 minutes) and washed in storage buffer twice and stored at 4 °C in 100-µL storage buffer. As controls uncoupled beads (without protein incubation) and L-selectin-coupled beads were generated using recombinant human L-selectin (R&D Systems).

Magnefy magnetic beads (1 µm, Bangs Laboratories) with COOH groups on the surface were washed 3 times in MiliQ water (1 ml) according to the manufacturer to remove contaminants in the storage buffer (0.05% NaN₃ in either de-ionized water or 5 mM Tris, 150 mM NaCl, Bangs Laboratory) and to reduce bead aggregation. Washed beads were incubated with 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDAC; PolyLink Protein Coupling Kit; Polysciences) in 200 μL for 15 min on a rotator at rt, followed by two wash steps using 200 μL coupling buffer as recommended to reduce bead aggregation. Beads were resuspended into 200 μL coupling buffer with each of the rSpTrf proteins or BSA (0.5 µM in 100 μL) for a total volume of 300 μL and incubated on a rotator at rt for 1 hour. Storage buffer (500 μL) was added and incubated with rotation at rt for 5 min to fill any remaining active sites on the beads with BSA. The beads were washed twice in 200 μL storage buffer, resuspended in 500 μL storage buffer, and stored at 4°C until used. The level of bead aggregation and protein binding was assessed by microscopy using rabbit-anti-V5-549 antibodies against the V5 tag on the rSpTrf proteins cross-linked to the beads. All wash steps were carried out with a magnet stand to collect the beads prior to removing the wash buffers.

Both preimmune mouse serum and anti-Sca polyclonal mouse serum (produced from Balb/c mice immunized with purified Sca proteins; Cosmogenetech, Seoul, South Korea) were used for the experiments. Human sera were prepared from scrub typhus patients following institutional review board approval. Horseradish peroxidase (HRP)-conjugated anti-mouse or anti-human IgG secondary antibodies (Santa Cruz Biotech Inc., Santa Cruz, CA) were used for immunoblotting [32 (link)]. The Alexa Fluor 488- or Alexa Fluor 594-conjugated anti-mouse, and-human antibodies used in the immunofluorescence assays were purchased from Molecular Probes (Invitrogen). For the beadbinding assay, Fluoresbrite microparticles (1 μm; Polyscience Inc., Warrington, PA) containing rhodamine were conjugated to GST or GST-ScaA proteins by using a PolyLink protein coupling kit (Polyscience Inc.) in accordance with the manufacturer’s instructions.

Carboxyl polystyrene beads (CP-20–10, diameter 2.1 μm, Spherotech) were covalently coated with sheep anti-digoxigenin antibody (Roche) via carbodiimide reaction (PolyLink Protein coupling kit, Polysciences, Inc.). Approximately 50 ng of the generated construct were incubated with 2 μL beads in 10 μL HMK buffer (50 mM HEPES, pH 7.5, 5 mM MgCl₂, 100 mM KCl) for 15 min in a rotary mixer at 4 °C and rediluted in 350 μL HMK buffer. With our coupling strategy, ~50% of the constructs will be asymmetrically functionalized with digoxigenin and biotin in each side. To create the second connection, we employed Neutravidin-coated polystyrene beads (NVP-20–5, diameter 2.1 μm, Spherotech). Once trapped, beads were brought into close proximity to allow binding and tether formation was identified by an increase in force when the beads were moved apart. To mitigate photobleaching and tether damage, we added an oxygen scavenging system (3 units/mL pyranose oxidase, 90 units/mL catalase, and 50 mM glucose, all purchased from Sigma-Aldrich).

To prepare beads with adsorbed antigen, a total of 130 × 10⁶ carboxylated latex beads of 1 μm diameter (Polysciences) were incubated overnight with a concentration of 40 μg/ml of protein in 1 ml of PBS at 4 °C. When more than one protein was adsorbed, equimolar amounts were used. Beads were subsequently washed twice with PBS plus 1% BSA and 2 mM EDTA, and resuspended in RPMI medium. To prepare beads with covalently-bound antigen, the PolyLink Protein Coupling Kit (Polysciences) was used as indicated by the manufacturer. Ovalbumin (OVA) and hen-egg lysozyme (HEL) were purchased from SIGMA; NIP(15)-OVA and NP(25)-CGG from Biosearch Technology.