Nhs biotin

Porcine brain tubulin was purified and covalently labelled with NHS-biotin (Thermo Fisher) or NHS-CF640R (Sigma-Aldrich) using standard procedures (Castoldi and Popov, 2003 (link), Hyman et al., 1991 (link)). CF640R-tubulin was labelled at a ratio of 0.4 fluorophores per tubulin dimer.

Surface biotinylation was performed on primary cultured neurons and CHO cells following established protocols^{48 (link)}. Cells were incubated in pH 7.4 or pH 6.0 solution for 1 h. After washing three times with an ice-cold PBS+/+ (PBS plus 1 mM MgCl₂ and 2.5 mM CaCl₂) solution, cells were incubated with 0.25 mg/ml NHS-biotin (21335, Thermo Scientific) in the PBS+/+ solution at 4 °C for 30 min, and then washed with the PBS+/+ solution containing 0.1 M glycine to quench the reaction. Ten percent volume of the lysate was saved for determination of total (T) proteins and mixed with 4× loading buffer. The remainder, representing surface (S) proteins, was isolated by NeutrAvidin Agarose Resin (TD264106, Thermo Scientific) and eluted as described above for co-immunoprecipitation.

Adapter molecules were either commercially available (Miltenyi Biotec, Bergisch Gladbach, Germany) or were generated from IgG antibodies with the f(ab)₂ or fab preparation kit following the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). Generated f(ab)₂s and fabs were labeled using NHS-Biotin (Thermo Fisher Scientific, Waltham, MA, USA) [24 ].

Imidazole, magnesium chloride (MgCl₂), potassium chloride (KCl), calcium chloride (CaCl₂), sodium azide (NaN₃), boric acid (H₃BO₃), monopotassium phosphate (KH₂PO₄), dimethyl sulfoxide (DMSO), tris-(hydroxymethyl)-aminomethan (Tris), dithiothreitol (DTT), adenosine triphosphate (ATP), sucrose, glucose, pyranose-oxidase (PO), catalase (C), creatine phosphokinase (CPK), creatine phosphate (CPH), Poly(ethylene glycol) 35 kDa (PEG), methyl cellulose 40 kDa (MC), decane, silicon oil 50 cSt, mineral oil, and chloroform were purchased from Sigma-Aldrich. Alexa Fluor 488 phalloidin (488 phalloidin), phalloidin, and NHS-Biotin were purchased from Thermo Fisher Scientific. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG(2000) Biotin) were purchased from Avanti Polar Lipids.

HUVEC were serum starved and stimulated with VEGF where indicated. All following steps were performed at 4 °C. Cells were washed with complete PBS (Sigma-Aldrich) and incubated 0.2 mg/ml NHS-Biotin (ThermoFisher Scientific) in complete PBS for 30 min. The unreacted biotinylation reagent was quenched by incubation with 100 mM glycine. Cells were then solubilized in lysis buffer (20 mM Tris pH 7.5, 137 mM NaCl, 1 mM EDTA, 1% NP40, 2 mM sodium orthovanadate, 10 mM sodium fluoride) containing protease inhibitor cocktail (Sigma-Aldrich). Cell lysates were centrifuged at 12,000 × g for 12 min at 4 °C and a sample was taken from the supernatant, which represented the total cellular protein. Streptavidin-agarose beads (Upstate Biotechnology, Lake Placid, NY) were added to the remaining supernatant (20 µl packed beads per 500 µl lysate) and left to tumble at 4 °C for 1 h. Beads were collected by centrifugation at 800 × g for 30 s at 4 °C and supernatant was removed—this sample represents the internal protein pool. The beads were then washed 3 times in lysis buffer at 4 °C and protein was extracted from the beads by heating at 95 °C with SDS-PAGE sample buffer—this represents the surface pool. Equivalent volumes of all three samples were resolved by SDS-PAGE and analyzed by western blotting.

To observe cell membrane deformation at MHz frequencies we used a Shimadzu HPV-X2 camera. Samples were illuminated using a 2W 532 nm laser (CNI, MLL-F-532-2W) controlled by an optical beam shutter (Thorlabs SH05, KSC101). Right-angle prism mirrors directed the laser light through a water bath and into a sample dish containing the imaged neurons. The transducer was positioned in the water tank at an angle of 45° relative to the water surface to minimize standing waves. 10× and 40× water immersion objectives (Leica, NA 0.3, Olympus, NA 0.8) were used. A series of prism mirrors and converging lenses with focal lengths of 200 mm and 50 mm delivered the image into the camera, which acquired 256 images over 51.2 µs. Images were acquired starting 100 ms after the onset of ultrasound stimulation, to capture events coincident with the initiation of calcium and voltage signals. As a positive control for detecting large scale deformation, a PDL-coated mylar film dish was biotinylated by incubating NHS-biotin (Thermo Fisher Scientific, 200 4 µg/ml) for 3 h. After washing the free linkers with PBS, the dish was then inverted and incubated for 1 h with streptavidin-functionalized microbubbles (Advanced Microbubbles Laboratories LLC SIMB3-4SA, 4 µm in diameter) to attach the bubbles onto the mylar film, and bubble cavitation was imaged using the same parameters as used with neurons.

Transfected M‐1 CCD cells in 10 cm plates were biotinylated following a previously published method (Hardege et al. 2015). Briefly, M‐1 CCD cells in 10 cm plates were washed once in NES and placed on ice. Cells were incubated in 0.2 mg/mL NHS‐ biotin ( www.thermofisher.com) for 45 min on ice before quenching with Tris buffer (25 mmol/L Tris‐HCl; 150 mmol/L NaCl; 10 mmol/L EDTA, pH 7.4). Cells were removed from the plate and centrifuged at 200 g for 5 min at 4°C. Supernatant was discarded and cells incubated in solubilization buffer (25 mmol/L Tris‐HCl; 150 mmol/L NaCl; 10 mmol/L EDTA; 1% Triton X‐100, pH7.4) with protease inhibitors ( www.roche.co.uk) at 4°C with agitation for 1 h. The resulting lysate was cleared by centrifugation at 18,000 g rpm 4^°C for 1 h. 30 μL of streptavidin beads ( www.thermofisher.com) per sample was washed once in wash buffer (25 mmol/L Tris‐HCl; 150 mmol/L NaCl; 10 mmol/L EDTA; 1% Triton X‐100, pH 7.4), centrifuged at 1800 g for 5 min at 4°C and supernatant removed. The supernatant was added to streptavidin beads and incubated with agitation for 2 h at 4°C. Beads were then pelleted at 18,000 g for 5 min at 4°C and washed three times in wash buffer before resuspending the pellet in sample buffer ( www.lifetechnologies.com).