White OptiPlate-96 (Perkin-Elmer, Norwalk, Connecticut, USA) microplates were coated with 10 μg/mL of nucleolin, RRM, RRM1, RRM2, RRM3, RRM4, GST, or OVA in carbonate buffer overnight at 4 °C. The coated plates were washed with PBS containing 0.05% Tween-20 and incubated with PBS containing 1% BSA for 1 h at 25 °C. After washing, 0.2 μM ODN1826, CpG K3, GpC K3, CpG K3(O), CpG D35, ODN1585, GpC D35, CRO or AS1411; or 2 μg/mL poly(I:C), all labeled with biotin, were incubated for 2 h at 25 °C, then with PE-labeled streptavidin (BioLegend) for 1 h at 25 °C. In the other experiment, the plates were incubated with 50 μg/mL PE-labeled MS-3 or PE-labeled isotype control antibody for 2 h at 25 °C. After washing the plates, fluorescence was measured using a multi-plate reader (DS Pharma Biomedical, Osaka, Japan).
Pe labeled streptavidin
Manufactured by BioLegend
PE-labeled streptavidin is a protein that binds to the biotinylated molecules. It is conjugated with the fluorescent dye phycoerythrin (PE), which allows detection and visualization of the labeled molecules.
Automatically generated - may contain errors
Lab products found in correlation
White optiplate 96, by PerkinElmer (1 mentions)
Multi plate reader, by Sumitomo Pharma (1 mentions)
Biotin labeled anti rat igg, by Jackson ImmunoResearch (1 mentions)
Fluosave reagent, by Merck Group (1 mentions)
Fluoview fv1000 inverted confocal microscope, by Olympus (1 mentions)
Pe labeled streptavidin, by Jackson ImmunoResearch (1 mentions)
4 protocols using pe labeled streptavidin
1
Nucleolin Binding Assay with ODNs
2
In Vivo Antigen Acquisition Protocol
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To study in vivo antigen acquisition, we adapted a protocol to generate HEL-containing immune complexes as described previously (Phan et al., 2007 (link)). CD45.1 mice were passively immunized with a polyclonal anti-PE antibody (Rockland Immunochemicals). The next day, 10 μg of biotinylated HEL or HEL3x protein, produced in house as described (Paus et al., 2006 (link)), was complexed to PE-labeled streptavidin (BioLegend) and injected s.c. near the inguinal and axillary LNs under isoflurane anesthesia. The following day, CD23Cre+Myh9fl/wtSWHEL and CD23Cre+Myh9fl/flSWHEL B cells were isolated, fluorescently labeled, mixed, and transferred to immunized recipient mice by i.v. injection. After 14 hr, inguinal and axillary LNs were harvested and analyzed by flow cytometry.
In vitro internalization and presentation of soluble antigen are described inSupplemental Experimental Procedures .
In vitro internalization and presentation of soluble antigen are described in
3
Single-cell Analysis of GPCR Complexes
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For single-cell analysis of 2PK-3 cells expressing Myc-tagged β2ARs together with Flag-tagged CCR7, CXCR4, or S1PR1, cells were stained with Alexa Fluor 647–conjugated anti-Myc (9B11; Cell Signaling Technology) and biotin-labeled antibodies against CCR7 (4B12) or CXCR4 (2B11), followed by PE-labeled streptavidin (BioLegend). S1PR1 was detected with a purified rat antibody (713412; R&D Systems), followed by biotin-labeled anti–rat IgG (Jackson ImmunoResearch Laboratories) and PE-labeled streptavidin (Arnon et al., 2011 (link)). Labeled cells were seeded onto slides and fixed with 4% paraformaldehyde. Slides were mounted with FluoSave Reagent (EMD Millipore). Images were acquired at room temperature with Fluoview FV10-ASW version 3.00 using a Fluoview FV1000 inverted confocal microscope (Olympus), equipped with plan apochromat UPLSAPO objectives (100× oil immersion with 1.40 NA). Co-localization of fluorescent signals was analyzed with a plug-in ‘RG2B Colocalization’ for ImageJ software.
4
HCV-specific CD8+ T cell Tetramer Staining
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