Ez link sulfo nhs biotinylation kit

Antibody reactivity to HES and hydrogen fluoride (HF)-treated HES was performed as described previously (Hewitson et al., 2011 (link)). For competition ELISA, plate bound HES was first incubated for 30 min at 37 °C with 250 μg/ml of unlabelled mAb before addition of 10 μg/ml of biotin-labelled mAb for 2 h at 37 °C. Antibodies were biotinylated with a 20-fold molar excess of biotin using EZ-link Sulfo-NHS Biotinylation kit (Thermo Fisher Scientific, USA). Bound biotinylated antibody was detected with 1/1000 streptavidin-horseradish peroxidase (Sigma–Aldrich, USA) and developed as described by Hewitson et al. (2011) (link).

NCM460 cells maintained for 14 days in RF deficient and OS media were biotinylated using an EZ-Link Sulfo-NHS-Biotinylation Kit (Thermo Fisher Scientific Inc., Rockford, IL) and then lysed. In order to isolate biotinylated proteins, an equal amount of total soluble protein from NCM460 cells maintained in RF deficient or OS media was then incubated with avidin-conjugated agarose. The agarose-avidin beads were washed three times following manufacturer’s protocol and the bound biotinylated protein eluted by heating with DTT at 95°C. The eluted protein samples were subjected to western blot analysis in a NuPAGE 4–12% Bis-Tris gradient minigel (Invitrogen). For normalization, an equal protein concentration of total NCM460 cell lysate was loaded simultaneously and the level of surface expression of hRFVT-3 was determined using hRFVT-3 specific primary antibodies. After washing with PBST the blots were probed with IRDye 800 labeled goat-anti rabbit (1:30,000) secondary antibody. Specific bands for hRFVT-3 were detected and quantified as described previously.

Purified α_vβ₃ receptors (Sino Biological Europe GmbH, Eschborn, Germany) were diluted to 0.5 μg/mL in coating buffer containing Tris-HCl (20 mmol/L; pH 7.4), NaCl (150 mmol/L), MnCl₂ (1 mmol/L), CaCl₂ (2 mmol/L) and MgCl₂ (1 mmol/L). An aliquot of diluted receptors (100 μL per well) was added to 96-well microtiter plates (MW 96F Medisorp Straight Nunc, Thermo Fischer Scientific, Waltham, MA, USA) and incubated, overnight, at 4 °C. The plates were then incubated with blocking solution (coating buffer plus 5% bovine serum albumin) for an additional 2 h at room temperature to block nonspecific binding followed by 3 h incubation at room temperature with various concentrations of test compounds in the presence of vitronectine (1 μg/mL, Sigma Aldrich, Merck KGaA, Darmstadt, Germany) biotinylated by using EZ-Link Sulfo-NHS-Biotinylation kit (Thermo Fisher, Waltham, MA, USA). After washing, the plates were incubated for 1 h at room temperature with streptavidin–biotinylated peroxidase complex (GE Healthcare, Chicago, IL, USA), followed by 30 min incubation with substrate reagent solution (100 μL; R&D Systems) before stopping the reaction by addition of H₂SO₄ (2 N, 50 μL). Absorbance at 415 nm was read with a BMG Labtech Fluostar Optima microplate reader. All the experiments were performed in triplicates and data collected were analyzed using the GraphPad 5.0 Software Package.

Purified SubB_{ΔS106/ΔT107} was labelled with biotin using the EZ-Link^® Sulfo-NHS-Biotinylation Kit (Thermo Scientific) according to the manufacturer’s instructions. Purified human and bovine α−1 acid glycoprotein (Sigma cat. nos G9885 and G3643) were dissolved in water at 5 mg/ml and 5 μl volumes of serial two-fold dilutions were spotted onto nitrocellulose filters and air dried at 37 °C overnight. Filters were then blocked with 5% skim milk in Tris-buffered saline with 0.05% Tween 20 (TTBS) for 2 h. After washing three times in TTBS, filters were overlaid with 1 μg/ml biotin-SubB_{ΔS106/ΔT107} in TTBS and incubated overnight at 4 °C. Filters were then washed three times in TTBS and bound biotin-SubB_{ΔS106/ΔT107} was detected using streptavidin-alkaline phosphatase conjugate (Roche). Filters were developed using a chromogenic nitro-blue tetrazolium/X-phosphate substrate system (Roche).

PBMC were isolated via a standard Ficoll gradient. Informed consent in writing was obtained from each donor. B cells were enriched from PBMC using Dynabeads Untouched Human B cells Kit (Thermo Fisher). For further analysis, B cells were incubated with human Fc block (BD Biosciences) for 10’ at RT, followed by biotinylated HBsAg for 30’ on ice (HBsAg by Roche Diagnostics; EZ-Link Sulfo-NHS-Biotinylation kit, Thermo Fisher). Memory B cell staining: 30’ on ice in PBS+0.5% BSA with live-dead stain (Thermo Fisher), anti-CD19-eF450 (eBioscience), anti-IgG-Pe-Cy7 (BD) and Streptavidin-APC (eBioscience). Cells were analyzed via flow cytometry on Cytoflex S (Beckman Coulter) or sorted with MoFlo II (Beckman Coulter) into lysis buffer, i.e. PBS+12U RNasin (Promega), 100 mM DTT (Thermo Fisher). PCR plates were sealed and stored at -80°C. IgG gene identification via PCR and sequencing from single B cells was performed as described elsewhere (16 (link)).