Biotin labeling kit nh2

Interaction between PfRipr and 13 erythrocyte surface proteins was quantified by AlphaScreen as reported^{40 (link)}. Briefly, reactions were carried out in 20 µl of reaction volume per well in 384-well OptiPlate microtiter plates (PerkinElmer). First, affinity-purified Ecto-PfRipr recombinant protein was biotinylated using a Biotin Labeling Kit-NH₂ (Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer’s instruction. Secondly, 5 µl of 10 nM biotinylated protein was mixed with 5 µl of 10 nM for each erythrocyte surface protein in reaction buffer (100 mM Tris-HCL [pH 8.0], 0.01% [v/v] Tween-20 and 0.1 mg/ml [w/v] bovine serum albumin), and incubated for 1 h at 26 °C to form a protein-protein complex. Subsequently, a 10 µl suspension of streptavidin-coated donor-beads and anti-GST acceptor-beads (PerkinElmer) mixture in 1:1 (v/v) in the reaction buffer was added to a final concentration of 15 µg/ml of both beads. The mixture was incubated at 26 °C for 12 h in the dark to allow the donor- and acceptor-beads to optimally bind to biotin and GST, respectively. Upon illumination of this complex, a luminescence signal at 620 nm was detected by the EnVision plate reader (PerkinElmer) and the results were expressed as AlphaScreen counts. GST tagged Rh5, known to interact with PfRipr, was included as a positive control and His-GST as a negative control.

Anti-CSE1L (H2) antibody-coated 96-well plates (Costar) were blocked with 5% BSA in TBS (Tris-buffered saline) for 1 h. The wells were washed with TBST (0.05% Tween 20 in TBS) and then incubated with serum samples (sixfold dilution with TBS) for 1 h. After being washed with TBST, the wells were incubated with biotin-conjugated anti-phospho-CSE1L antibodies for 1 h. The biotin-conjugated anti-phospho-CSE1L antibodies were prepared by biotinylating anti-phospho-CSE1L antibodies using the Biotin Labeling Kit-NH2 (Dojindo Laboratories, Kumamoto, Japan). The wells were then washed with TBST, reacted with streptavidin-conjugated horseradish peroxidase (R&D Systems, Minneapolis, MN, USA), and followed by incubation with the substrate reagent (R&D Systems). For calibration, two blank wells containing TBST were used as the background value, and two wells that were not coated with anti-phospho-CSE1L antibodies but reacted with all other ELISA reagents were used as control wells. The absorbance at 450 nm was measured within 20 min following the reaction by using a Thermo Multiskan EX microplate photometer (Thermo Fisher Scientific, Waltham, MA, USA). Each sample was assayed two times.

We aimed to confirm that purified αS1‐casein was bound to cell surface sIgE and activated the PS‐basophils. Purified αS1‐casein was labeled with biotin using the Biotin Labeling Kit‐NH2 (Dojindo Molecular Technologies, Kumamoto, Japan). Biotin‐labeled αS1‐casein was added to PS‐basophil suspension and incubated on ice for 30 minutes. After washing, anti‐human IgE‐PE (BioLegend, San Diego, CA), CRA1 (anti‐human FcεRIα‐PE/Cy7, detecting non‐IgE‐binding site on FcεRIα; BioLegend), CRA2 (anti‐human FcεRIα‐FITC, detecting IgE‐binding site on FcεRIα; BioAcademia, Osaka, Japan), anti‐human CD3‐APC/Alexa Fluor 750 (Beckman Coulter), anti‐human CRTH2‐PerCP/Cy5.5 (BioLegend), and streptavidin‐Brilliant violet 421 (BioLegend) were added. The reaction was allowed to proceed for 30 minutes in the dark at 4°C and was stopped by adding the stop solution. After adding Fix and Lyse solution and washing, cells were resuspended in 0.5 mL PBS containing 0.1% formaldehyde and analyzed using the flow cytometer.

Two monoclonal antibodies (mAb2H6 and mAb10D11) specific for r-S100A8 and r-S100A9, respectively, were used to establish an ELISA for the heterodimer, in which the two mAbs were provided from Yamasa Corporation (Chiba, Japan). Horseradish Peroxidase (HRP) Labeling Kit-SH and Biotin Labeling Kit-NH2 were purchased from DOJINDO LABORATORIES (Kumamoto, Japan). ELISA kits for inflammatory biomarkers, such as CRP, IL-6, TNF-α, and IL-1β, were purchased from COSMO BIO Co. Ltd. (Tokyo, Japan) to quantitatively determine their serum concentrations in rats. Dextran sulfate sodium salt (DSS, MW: 36000–50000, MP Biochemicals) was purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). Tacrolimus was provided by Astellas Pharmacology, Inc (Tokyo, Japan). Clinical Thioglycollate Medium (E-MC17) was obtained from EIKEN Chemical Co. Ltd. (Tokyo, Japan). LPS from Escherichia coli (E. coli) was purchased from Sigma-Aldrich Co. LLC (Tokyo, Japan). VECTASHIELD Mounting Medium containing 4′,6-Diamidino-2-phenylindole, dihydrochloride (DAPI), streptavidin (STA)-fluorescein 5-isothiocyanate (FITC); and anti-mouse IgG (horse)-Texas Red (TR) conjugates were obtained from Vector Inc. (Burlingame, CA). All other reagents were obtained from Wakenyaku Co. Ltd. (Kyoto, Japan).