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glycosylation assay  (New England Biolabs)
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New England Biolabs glycosylation assay
a , Flow cytometric analysis showing the expression levels of SIGN-R1 in tracheal DC and RTMϕ at day 3 p.i. (n = 5 mice per group). b , Representative scatterplots showing the percentage of SIGN-R1+ IDC in trachea at day 3 p.i.. c , Representative 3D reconstruction of confocal micrographs showing the colocalisation between DIO-labelled PR8 (PR8-DIO) and SIGN-R1 (white arrows), expressed in an IDC one hour after in vitro culture. Bar indicates 2 μm (left) and 0.5 μm (right). d , Sensogramms of Surface Plasmon Resonance (SPR) analysis of the HA-binding affinity of SIGN-R1 Fc chimera protein (SIGN-R1-Fc.) SIGN-R1-Fc with or without <t>glycosylation</t> (after treatment with N-glycosidase F (PNGase F)), with different concentration of H1, below their respective kinetic parameters, calculated by SPR using purified proteins are shown. N.B., no binding detected. e , ELISA analysis of the HA-binding titre from different subtypes of the recombinant mouse SIGN-R1-Fc (n = 2 replicates per group). f , (Left) Representative scatterplots from flow cytometric analysis showing the capture of PR8-DIO by SIGN-R1+ IDC after an incubation period of 1 h with the virus. In the αSIGN-R1 group, the binding was inhibited by administration of a blocking antibody previous to the incubation with the virus. (Right) Mean fluorescence intensity (MFI) analysis showing differences in the capture of PR8-DIO by IDC treated with αSIGN-R1 blocking antibody with respect to the control groups (n = 4, 4 and 3 replicates per respective group). The presented data are representative of at least three ( a-c and f ) or two ( d-e ) independent experiments. Results are given as mean ± SD ( a , b and f ), as mean± SEM ( d ) or mean alone ( e ).
Glycosylation Assay, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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a , Flow cytometric analysis showing the expression levels of SIGN-R1 in tracheal DC and RTMϕ at day 3 p.i. (n = 5 mice per group). b , Representative scatterplots showing the percentage of SIGN-R1+ IDC in trachea at day 3 p.i.. c , Representative 3D reconstruction of confocal micrographs showing the colocalisation between DIO-labelled PR8 (PR8-DIO) and SIGN-R1 (white arrows), expressed in an IDC one hour after in vitro culture. Bar indicates 2 μm (left) and 0.5 μm (right). d , Sensogramms of Surface Plasmon Resonance (SPR) analysis of the HA-binding affinity of SIGN-R1 Fc chimera protein (SIGN-R1-Fc.) SIGN-R1-Fc with or without glycosylation (after treatment with N-glycosidase F (PNGase F)), with different concentration of H1, below their respective kinetic parameters, calculated by SPR using purified proteins are shown. N.B., no binding detected. e , ELISA analysis of the HA-binding titre from different subtypes of the recombinant mouse SIGN-R1-Fc (n = 2 replicates per group). f , (Left) Representative scatterplots from flow cytometric analysis showing the capture of PR8-DIO by SIGN-R1+ IDC after an incubation period of 1 h with the virus. In the αSIGN-R1 group, the binding was inhibited by administration of a blocking antibody previous to the incubation with the virus. (Right) Mean fluorescence intensity (MFI) analysis showing differences in the capture of PR8-DIO by IDC treated with αSIGN-R1 blocking antibody with respect to the control groups (n = 4, 4 and 3 replicates per respective group). The presented data are representative of at least three ( a-c and f ) or two ( d-e ) independent experiments. Results are given as mean ± SD ( a , b and f ), as mean± SEM ( d ) or mean alone ( e ).

Journal: Nature microbiology

Article Title: Protection against influenza infection requires early recognition by inflammatory dendritic cells through C-type lectin receptor SIGN-R1

doi: 10.1038/s41564-019-0506-6

Figure Lengend Snippet: a , Flow cytometric analysis showing the expression levels of SIGN-R1 in tracheal DC and RTMϕ at day 3 p.i. (n = 5 mice per group). b , Representative scatterplots showing the percentage of SIGN-R1+ IDC in trachea at day 3 p.i.. c , Representative 3D reconstruction of confocal micrographs showing the colocalisation between DIO-labelled PR8 (PR8-DIO) and SIGN-R1 (white arrows), expressed in an IDC one hour after in vitro culture. Bar indicates 2 μm (left) and 0.5 μm (right). d , Sensogramms of Surface Plasmon Resonance (SPR) analysis of the HA-binding affinity of SIGN-R1 Fc chimera protein (SIGN-R1-Fc.) SIGN-R1-Fc with or without glycosylation (after treatment with N-glycosidase F (PNGase F)), with different concentration of H1, below their respective kinetic parameters, calculated by SPR using purified proteins are shown. N.B., no binding detected. e , ELISA analysis of the HA-binding titre from different subtypes of the recombinant mouse SIGN-R1-Fc (n = 2 replicates per group). f , (Left) Representative scatterplots from flow cytometric analysis showing the capture of PR8-DIO by SIGN-R1+ IDC after an incubation period of 1 h with the virus. In the αSIGN-R1 group, the binding was inhibited by administration of a blocking antibody previous to the incubation with the virus. (Right) Mean fluorescence intensity (MFI) analysis showing differences in the capture of PR8-DIO by IDC treated with αSIGN-R1 blocking antibody with respect to the control groups (n = 4, 4 and 3 replicates per respective group). The presented data are representative of at least three ( a-c and f ) or two ( d-e ) independent experiments. Results are given as mean ± SD ( a , b and f ), as mean± SEM ( d ) or mean alone ( e ).

Article Snippet: Hemagglutinin HA from Influenza A H1N1 (A/Puerto Rico/8/34) was used for glycosylation assay using Endo Hf (NEB) or PNGase F (NEB) using the manufacturer’s instructions.

Techniques: Expressing, In Vitro, SPR Assay, Binding Assay, Concentration Assay, Purification, Enzyme-linked Immunosorbent Assay, Recombinant, Incubation, Blocking Assay, Fluorescence