anti human il 15rα polyclonal goat igg antibodies  (R&D Systems)

Journal: Glycoconjugate journal

Article Title: Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N - and O -linked glycosylation

doi: 10.1007/s10719-015-9627-1

Figure Lengend Snippet: Overview of the full length and mature amino acid sequences and numbering (subscript) and the identified N - and O -glycosylation sites (occupied sites in bold, red; unoccupied sites in bold, black) of human IL-15 and sIL-15Rα. The same amino acid sequences of IL-15 and sIL-15Rα were used for the two large-scale preparations of hetIL-15 ( i.e. the cGMP and EN lots)

Article Snippet: The identity of IL-15 and sIL-15Rα was confirmed by Western immunoblotting using anti-human IL-15 or anti-human IL-15Rα polyclonal goat IgG antibodies (product #AF315 and #AF247, respectively, R&D Systems).

Techniques:

Journal: Glycoconjugate journal

Article Title: Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N - and O -linked glycosylation

doi: 10.1007/s10719-015-9627-1

Figure Lengend Snippet: Overview of the expression, cellular presentation, function and analysis of the soluble human hetIL-15 complex. a. Engineered human IL-15 and IL-15Rα were co-expressed and secreted by HEK293 cells as a soluble heterodimeric complex (hetIL-15) after proteolytic cleavage from the cell surface. The complex binds to the IL-2Rβ/γ receptor complex located on target cells, where it initiates a cellular response. b. The hetIL-15 complex was isolated and its purity monitored using reducing (upper) and native (lower) SDS-PAGE with Coomassie blue staining (left gels) and Western blotting using anti-IL-15 and anti-IL-15Rα antibodies (right gels). The two clinically relevant preparations of hetIL-15 (i.e. the EN and cGMP lots) are shown. c. IL-15 and sIL-15Rα were separated from their heterodimeric complex by non-reductive RP-HPLC and individually subjected to LC-MS/MS-based glycan (top), glycopeptide (bottom) and glycoprotein (right) profiling in order to characterize their N- and O-glycosylation in a detailed and site-specific manner

Article Snippet: The identity of IL-15 and sIL-15Rα was confirmed by Western immunoblotting using anti-human IL-15 or anti-human IL-15Rα polyclonal goat IgG antibodies (product #AF315 and #AF247, respectively, R&D Systems).

Techniques: Expressing, Isolation, SDS Page, Staining, Western Blot, Liquid Chromatography with Mass Spectroscopy

Journal: Glycoconjugate journal

Article Title: Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N - and O -linked glycosylation

doi: 10.1007/s10719-015-9627-1

Figure Lengend Snippet: Glycome profiling demonstrates extensive and reproducible N-and O-glycosylation of IL-15 and sIL-15Rα in the large-scale preparations of hetIL-15 (cGMP and EN lots). N-glycans of IL-15 (a) and sIL-15Rα (b) were structurally characterized and quantitatively profiled using PGC-LC-ESI-negative ion-CID-MS/MS. c. Several isobaric N-glycan isomers were identified as exemplified by the extracted ion chromatogram (EIC) of the abundant Man3GlcNAc5Fuc1 composition (m/z 832.9 [M – 2 H]2−, upper panel) and the corresponding CID-MS/MS (bottom panel, see Fig. 3 for key to monosaccharide symbols) demonstrating three isobaric GlcNAc-terminating N-glycan isomers i.e. N-glycan structure 5a (shown), 5b and 5c (fragment spectra for the two latter N-glycans are presented in Supplementary Fig. S1). ‘*’ represents a non-glycan signal interference. d. The O-glycome profiling of sIL-15Rα showed less micro-heterogeneity. *Structure 2a/2b could not be consistently separated and were thus combined for quantitation purposes. No O-glycosylation was detected for IL-15 (data not shown). The N- and O-glycosylation profiles of IL-15 and sIL-15Rα of the EN (red bars) and cGMP (blue bars) lots of hetIL-15 were similar as evaluated by their high correlation coefficients (R2 = 0.815–0.982). The relative glycan quantities are averages of technical duplicates (see Supplementary Table S1 and S2 for exact values). The corresponding N- and O-glycan structures and their biosynthetic relationship are depicted in Fig. 3

Article Snippet: The identity of IL-15 and sIL-15Rα was confirmed by Western immunoblotting using anti-human IL-15 or anti-human IL-15Rα polyclonal goat IgG antibodies (product #AF315 and #AF247, respectively, R&D Systems).

Techniques: Tandem Mass Spectroscopy, Quantitation Assay

Journal: Glycoconjugate journal

Article Title: Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N - and O -linked glycosylation

doi: 10.1007/s10719-015-9627-1

Figure Lengend Snippet: Structures and biosynthetic relationship of the observed IL-15 and sIL-15Rα N- and O-glycans. The designated numbers of the individual N-linked (left) and O-linked (right) glycans correspond to the numbering used in Fig. 2, Fig. 5 and Supplementary Tables S1–S2. Their biosynthetic interconnectivity is presented with arrows symbolizing single glycosylation enzyme reactions. The most abundant N- and O-glycans are shaded in dark grey. Monosaccharide symbols are presented according to the Essentials of Glycobiology/Consortium for Functional Glycomics nomenclature. Key: fucose (red triangle), mannose (green circle), GlcNAc (blue square), sialic acid (NeuAc) (purple diamond), galactose (yellow circle) and HexNAc (unspecified GlcNAc or GalNAc) (open square)

Article Snippet: The identity of IL-15 and sIL-15Rα was confirmed by Western immunoblotting using anti-human IL-15 or anti-human IL-15Rα polyclonal goat IgG antibodies (product #AF315 and #AF247, respectively, R&D Systems).

Techniques: Functional Assay

Journal: Glycoconjugate journal

Article Title: Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N - and O -linked glycosylation

doi: 10.1007/s10719-015-9627-1

Figure Lengend Snippet: Site-specific O-glycoprofiling of sIL-15Rα of clinical-grade hetIL-15 (cGMP lot) using RP (C18)-LC-ESI-positive ion-CID/ETD-MS/MS. a. The MS1 level profile (right) indicated multiple Thr81- and Thr86 - glycoforms on the tryptic O - glycopeptide R - ] 74 PA P PAPPSTVTTAGVTPQPESLSPSGK97[−E. ETD and CID fragmentation (left spectra) confirmed the O-glycosylation sites and the structure of the two conjugated core 1-type O-glycansans (HexHexNAcNeuAc, corresponding to structure 2b, Fig. 3), m/z 905.4 (4+). Additional examples of ETD-MS/MS fragment spectra of two other sIL-15Rα tryptic O-glycopeptides i. e. b. The N-terminal-1ITCPPPMSVEHADIWVK17-[S peptide conjugated with a single HexNAc (corresponding to structure i, Fig. 3) m/z 728.9 (3+) and c. the C-terminal peptide K-]152NWELTASASHQPPGVYPQG170[−conjugated with two core 1-type O-sialoglycans (HexHexNAcNeuAc, corresponding to structure 2a or 2b, latter shown) and one core 1-type O-asialoglycan (HexHexNAc, structure ii, Fig. 3) m/z 930.8 (4+). Key fragment ions for exact site localization are presented in red. See Fig. 3 for monosaccharide key

Article Snippet: The identity of IL-15 and sIL-15Rα was confirmed by Western immunoblotting using anti-human IL-15 or anti-human IL-15Rα polyclonal goat IgG antibodies (product #AF315 and #AF247, respectively, R&D Systems).

Techniques: Tandem Mass Spectroscopy

Journal: Glycoconjugate journal

Article Title: Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N - and O -linked glycosylation

doi: 10.1007/s10719-015-9627-1

Figure Lengend Snippet: Overview of the identified sIL-15Rα tryptic O -glycopeptides. The modified amino acid residues are underlined where known or listed as ND where unknown. The theoretical glycopeptide masses are based on carbamidomethylated cysteine residues. For peptides where the non-glycosylated variants were observed, the non-enriched LC-MS/MS data were used to establish the relative site-occupancy; otherwise glycopeptide-enriched LC-MS/MS data were used to establish the relative glycoform distribution. See for examples of assigned ETD/CID-MS/MS O -glycopeptide spectra

Article Snippet: The identity of IL-15 and sIL-15Rα was confirmed by Western immunoblotting using anti-human IL-15 or anti-human IL-15Rα polyclonal goat IgG antibodies (product #AF315 and #AF247, respectively, R&D Systems).

Techniques: Modification, Tandem Mass Spectroscopy

Journal: Glycoconjugate journal

Article Title: Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N - and O -linked glycosylation

doi: 10.1007/s10719-015-9627-1

Figure Lengend Snippet: Spatial map of the N- and O-glycosylation sites of hetIL-15 in its quaternary complex with IL-2Rβ and IL-2Rγ. The three putative N-glycosylation sites of IL-15 (green) are shown in blue. Only Asn79 was found to be occupied; Asn71 and Asn112 found on the interface to IL-2Rβ (cyan) and IL-2Rγ (magenta), respectively, were not utilized as N-glycosylation sites when expressed in HEK293. The available crystal structure (PDB: 4GS7) covered only the lightly O-glycosylated N-terminal region of the sIL-15Rα polypeptide chain (yellow) [34]. The occupied O-glycosylation site at Thr2 covered by this region is shown in orange. See also Fig. 1a. for schematic illustration of the quaternary complex

Article Snippet: The identity of IL-15 and sIL-15Rα was confirmed by Western immunoblotting using anti-human IL-15 or anti-human IL-15Rα polyclonal goat IgG antibodies (product #AF315 and #AF247, respectively, R&D Systems).

Techniques: