Ez link sulfo nhs biotin

Human Magnetic Luminex Assays (LXSAHM-15, LXSAHM-08, and LXSAHM-02) were purchased from R&D Systems (Minneapolis, MN). Magnetic COOH beads, amine coupling kits, and Bio-Plex Pro Reagent kits were purchased from Bio-Rad Laboratories (Hercules, CA). NHS and Sulfo-NHS, EDC, EZ-Link^TM Sulfo-NHS-Biotin, and Zeba^TM Spin Desalting Columns were purchased from Thermo Scientific (Rockford, IL). Agarose bound Aleuria Aurantia Lectin (AAL) was purchased from Vector Laboratories (Burlingame, CA). Pierce™ BCA Protein Assay Kit was purchased from Thermo Fisher Scientific (Waltham, MA).

Cloning and purification of SARS-CoV-2 NP and spike RBD were performed as we described previously [9 (link)]. The purified NP and spike protein RBD were biotinylated with EZ-link^TM Sulfo-NHS-Biotin (ThermoFisher Scientific, Waltham, MA, USA). Please refer to Supplementary Methods for details.

The tracer experiment was performed to determine the structural integrity of the intestinal barrier, as described in our previous study [35 (link)]. EZ-link Sulfo-NHS-Biotin (Pierce Chemical, Rockford, IL, USA) was used as a molecular tracer, which was diluted to a concentration of 2 mg/mL with PBS (pH 7.4) plus 1 mM CaCl₂ before use. Briefly, a small section (about 2 cm) of jejunum and colon was isolated immediately after the mice were sacrificed, and placed in PBS (pH 7.4) at 37 °C. Then, the prepared biotin was added to the intestinal cavity gently, followed by ligation at both ends. The intestinal tissue samples were incubated at room temperature for 5 min, and then fixed with 4% paraformaldehyde (PFA) in PBS (pH 7.4) for 3 h. After being washed four times (5 min each) in ice-cold PBS (pH 7.4), the fixed tissue was cut into 5 μm slide sections via freezing microtome (Leica, Wetzlar, Germany) and then incubated with a 1:800 dilution of Alexa-Fluor-488-conjugated streptavidin for 30 min under the light-proof conditions. The distributions of biotin in intestinal epithelial cells were observed and photographed using a Zeiss microscope (Zeiss, Oberkochen, Germany).

EZ-link Sulfo-NHS-Biotin (Pierce Chemical, Rockford, IL) was specific to label the cell surface proteins, and it was not able to permeate the intact cell membrane. The working concentration of biotin was 2 mg/ml in PBS containing 1mmol/L CaCl₂. We ligated 1 cm middle part of ileum and colon after the animals were sacrificed. Biotin was slowly injected into the ligated parts and incubated for 5 min. Then the ligated parts were excised and placed into 4% paraformaldehyde in PBS (pH 7.3) for 3 h. After washing three times, 5 μm frozen tissue sections were made by freezing microtome (CM 1900, Leica Microsystems, Heidelberg GmbH, Mannheim, Germany). Finally, sections were incubated with streptavidin conjugated to Alexa 488 for 30 min [27 (link)].

Sections of the jejunum and colon were obtained from the middle and ligated at one end. Then, EZ-Link Sulfo-NHS-Biotin (Pierce Chemical, Rockford, IL) was slowly injected into the intestine samples from the other end, which was then ligated. The samples were incubated for 5 min at room temperature, then fixed in 4% paraformaldehyde for >3 h. After washing three times with ice-cold PBS, 5 μm thick frozen tissue sections were prepared and incubated with streptavidin conjugated to Alexa Fluor 488 for 30 min. Finally, the distribution of biotin in the intestine was observed and photographed with a BX53 fluorescence microscope (Olympus, Tokyo, Japan).

We performed a tracer experiment using a primary amine-reactive biotinylation reagent which labels proteins and primary amine-containing macromolecules on a cell surface [23 (link)]. Epithelia grown on 12-mm filters were incubated in solution A with 0.01mg/ml EZ-Link Sulfo-NHS-Biotin (Life Technologies) in the basal side for 10 min at 37°C. Then epithelia were fixed with 10% paraformaldehyde for 7 min and pemeabilized with Triton X-100. The bound biotin was detected by alexa fluor 555 streptavidin (S-32355; Life Technologies). The samples were imaged on a Zeiss LSM700 confocal microscope in a similar manner as described in Immunocytochemistry.