Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Protein-specific analysis of invertebrate glycoproteins

doi: 10.1007/978-1-4939-8814-3_24

Figure Lengend Snippet: List of selected antibodies, lectins and pentraxins for N-glycan epitope screening ( Note 4 )

Article Snippet: SDS-PAGE and Western blotting list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 12% SDS-PAGE gel (using 40% acrylamide stock from Bio-Rad, diluted with either stacking gel buffer with 0.5 M Tris/HCl pH 6.8 or separation gel buffer with 1.5 M Tris/HCl pH 8.8) SDS-PAGE running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS; components from either VWR or Roth) Western blotting transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol; VWR or Roth) SDS-PAGE protein standard ladder (e.g., Thermo PageRuler™) Nitrocellulose membrane (BioTrace™ NT from Pall Life Science) Extra thick blotting paper (Bio-Rad) 0.5 % (w/v) Ponceau S (Sigma) in 1 % (v/v) acetic acid solution Membrane washing buffer: Tris buffered saline (TBS, i.e., 0.1 M Tris/HCl, pH 7.4, 0.1 M NaCl; typically made as a 10-fold concentrated stock) with 0.05% Tween (Sigma) Membrane blocking and antibody/lectin dilution buffer: Tris buffered saline with 0.05% Tween and 0.5% BSA (Roth) Primary, secondary antibodies, lectins and pentraxins (Sigma or Vector Laboratories) (see ) table ft1 table-wrap mode="anchored" t5 caption a7 Antibody (1 st ) Dilution Epitope( 13 , 14 ) Supplier Anti-HRP from rabbit, 10 mg/ml 1:10000 Core α1,3-Fuc/ core β1,2-Xyl Sigma Anti-PC (TEPC-15 mouse IgA), 10 mg/ml 1:200 PC-Hex(NAc) Sigma Antibody (2 nd ) Anti-rabbit IgG from goat conjugated with alkaline phosphatase 1:2000 Vector Labs Anti-mouse IgA from goat conjugated with alkaline phosphatase 1:10000 Sigma Pentraxin C reactive Protein (CRP) from human plasma (CaCl 2 2.5 mM added) 1:200 PC-Hex(NAc) MP Biochemicals Amyloid P component from human serum (SAP) 1:200 PE-Hex(NAc) Sigma Pentraxin recognition Anti-human C reactive protein from rabbit 1:1000 Dako Anti-Amyloid P human serum component IgG from rabbit (anti SAP) 1:1000 Calbiochem Lectin Biotinylated Aleuria aurantia lectin 1:1000 Core α1,6-Fuc/Lex Vector Labs Biotinylated wheat germ agglutinin 1:1000 β1,4HexNAc/α2,3Sia Vector Labs Biotinylated peanut agglutinin 1:1000 Galβ1,3GalNAc Vector Labs Lectin recognition Anti-biotin from goat conjugated with alkaline phosphatase 1:10000 Sigma Open in a separate window caption a8 List of selected antibodies, lectins and pentraxins for N-glycan epitope screening ( Note 4 ) SigmaFAST BCIP/NBT or SigmaFAST 3,3´-diaminobenzidine tetrahydrochloride tablets (Sigma), dissolved in 10ml and 5ml respectively.

Techniques: Plasmid Preparation

Journal: Glycoconjugate Journal

Article Title: Changes in fucosylation of human seminal IgG and secretory component of IgA in leukocytospermic patients

doi: 10.1007/s10719-013-9501-y

Figure Lengend Snippet: Relative reactivity of seminal plasma IgG with fucose-specific lectins

Article Snippet: Three biotinylated fucose-specific lectins (Vector Laboratories Inc., Burlingame, CA, USA): Aleuria aurantia lectin (AAL), Ulex europaeus agglutinin (UEA) and Lotus tetragonolobus agglutinin (LTA) were used to determine expression of fucose moiety in IgG by lectin-ELISA according to the procedure described previously for fibronectin and α 1 -acid glycoprotein [ ].

Techniques:

Journal: Glycoconjugate Journal

Article Title: Changes in fucosylation of human seminal IgG and secretory component of IgA in leukocytospermic patients

doi: 10.1007/s10719-013-9501-y

Figure Lengend Snippet: IgG relative reactivity with fucose-specific lectins. The relative reactivity of seminal IgG with biotinylated fucose-specific lectins AAL, UEA and LTA was determined in normal and leukocytospermic groups using lectin-ELISA and expressed in absorbance units (AU). □ – median value

Article Snippet: Three biotinylated fucose-specific lectins (Vector Laboratories Inc., Burlingame, CA, USA): Aleuria aurantia lectin (AAL), Ulex europaeus agglutinin (UEA) and Lotus tetragonolobus agglutinin (LTA) were used to determine expression of fucose moiety in IgG by lectin-ELISA according to the procedure described previously for fibronectin and α 1 -acid glycoprotein [ ].

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Glycoconjugate Journal

Article Title: Changes in fucosylation of human seminal IgG and secretory component of IgA in leukocytospermic patients

doi: 10.1007/s10719-013-9501-y

Figure Lengend Snippet: Dependence between IgG concentration and IgG relative reactivity with lectins. Correlation between IgG concentration and IgG relative reactivity with AAL ( a ), and UEA ( b ) was estimated according to a Spearman test, with a two-tailed p -value of less than 0.05 considered significant. 95 % confidence interval is marked by dotted lines

Article Snippet: Three biotinylated fucose-specific lectins (Vector Laboratories Inc., Burlingame, CA, USA): Aleuria aurantia lectin (AAL), Ulex europaeus agglutinin (UEA) and Lotus tetragonolobus agglutinin (LTA) were used to determine expression of fucose moiety in IgG by lectin-ELISA according to the procedure described previously for fibronectin and α 1 -acid glycoprotein [ ].

Techniques: Concentration Assay, Two Tailed Test

Journal: Glycoconjugate Journal

Article Title: Changes in fucosylation of human seminal IgG and secretory component of IgA in leukocytospermic patients

doi: 10.1007/s10719-013-9501-y

Figure Lengend Snippet: Relative reactivity of SC with fucose-specific lectins. Lectin-blotting with three biotinylated fucose-specific lectins AAL, UEA and LTA was performed for normal ( 1 ) and leukocytospermic ( 2 ) seminal groups. The bands corresponding to SC (78 ± 4.2 kDa and 63 ± 2.3 kDa) are shown by arrows

Article Snippet: Three biotinylated fucose-specific lectins (Vector Laboratories Inc., Burlingame, CA, USA): Aleuria aurantia lectin (AAL), Ulex europaeus agglutinin (UEA) and Lotus tetragonolobus agglutinin (LTA) were used to determine expression of fucose moiety in IgG by lectin-ELISA according to the procedure described previously for fibronectin and α 1 -acid glycoprotein [ ].

Techniques:

Journal: Glycoconjugate Journal

Article Title: Changes in fucosylation of human seminal IgG and secretory component of IgA in leukocytospermic patients

doi: 10.1007/s10719-013-9501-y

Figure Lengend Snippet: IgG relative reactivity with fucose-specific lectins. The relative reactivity of seminal IgG with biotinylated fucose-specific lectins AAL, UEA and LTA was determined in normal and leukocytospermic groups using lectin-ELISA and expressed in absorbance units (AU). □ – median value

Article Snippet: Three biotinylated fucose-specific lectins (Vector Laboratories Inc., Burlingame, CA, USA): Aleuria aurantia lectin (AAL), Ulex europaeus agglutinin (UEA) and Lotus tetragonolobus agglutinin (LTA) were used to determine expression of fucose moiety in IgG by lectin-ELISA according to the procedure described previously for fibronectin and α 1 -acid glycoprotein [ ].

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Glycoconjugate Journal

Article Title: Changes in fucosylation of human seminal IgG and secretory component of IgA in leukocytospermic patients

doi: 10.1007/s10719-013-9501-y

Figure Lengend Snippet: Relative reactivity of SC with fucose-specific lectins. Lectin-blotting with three biotinylated fucose-specific lectins AAL, UEA and LTA was performed for normal ( 1 ) and leukocytospermic ( 2 ) seminal groups. The bands corresponding to SC (78 ± 4.2 kDa and 63 ± 2.3 kDa) are shown by arrows

Article Snippet: Three biotinylated fucose-specific lectins (Vector Laboratories Inc., Burlingame, CA, USA): Aleuria aurantia lectin (AAL), Ulex europaeus agglutinin (UEA) and Lotus tetragonolobus agglutinin (LTA) were used to determine expression of fucose moiety in IgG by lectin-ELISA according to the procedure described previously for fibronectin and α 1 -acid glycoprotein [ ].

Techniques:

Journal: Journal of Inflammation Research

Article Title: The Alterations of Serum IgG Fucosylation as a Potential Additional New Diagnostic Marker in Advanced Endometriosis

doi: 10.2147/JIR.S341906

Figure Lengend Snippet: Relative Reactivities of Serum Native IgG and Isolated Serum IgG Glycans with Fucose-Specific Lectins

Article Snippet: The method was based on the reactivity of IgG sugar moieties with four fucose-specific biotinylated lectins (Vector Laboratories Inc., Burlingame, CA, USA): 1) Aleuria aurantia lectin (AAL, recognizing fucose α1,6-linked to the N-acetylglucosamine core of N-glycans and with lower affinity fucoses α1,2-, α1,3- and α1,4-linked of the outer arms, 2) Lens culinaris agglutinin (LCA, recognizing sequences containing fucosylated tri-mannose N-glycan core sites), 3) Lotus tetragonolobus agglutinin (LTA, specifically reacting with fucose α1,3-linked to GlcNAc, characteristic for Lewis x oligosaccharide structures, however, it can also slightly react with fucose typical for Lewis a and Lewis y structures.

Techniques: Isolation

Journal: Journal of Inflammation Research

Article Title: The Alterations of Serum IgG Fucosylation as a Potential Additional New Diagnostic Marker in Advanced Endometriosis

doi: 10.2147/JIR.S341906

Figure Lengend Snippet: Relative reactivities of serum native IgG (s-IgG), and isolated serum IgG (i–IgG) glycans with fucose-specific lectins: AAL - Aleuria aurantia lectin, LCA - Lens culinaris agglutinin, LTA - Lotus tetragonolobus agglutinin, UEA - Ulex europaeus agglutinin ( A – D ). For lectins specificity see Materials and methods section. Significant differences versus groups: A E, B NE. Median is indicated as a square. A two-tailed p-value of less than 0.05 was considered as significant (*p < 0.05; ***p < 0.001).

Article Snippet: The method was based on the reactivity of IgG sugar moieties with four fucose-specific biotinylated lectins (Vector Laboratories Inc., Burlingame, CA, USA): 1) Aleuria aurantia lectin (AAL, recognizing fucose α1,6-linked to the N-acetylglucosamine core of N-glycans and with lower affinity fucoses α1,2-, α1,3- and α1,4-linked of the outer arms, 2) Lens culinaris agglutinin (LCA, recognizing sequences containing fucosylated tri-mannose N-glycan core sites), 3) Lotus tetragonolobus agglutinin (LTA, specifically reacting with fucose α1,3-linked to GlcNAc, characteristic for Lewis x oligosaccharide structures, however, it can also slightly react with fucose typical for Lewis a and Lewis y structures.

Techniques: Isolation, Two Tailed Test

Journal: Journal of Inflammation Research

Article Title: The Alterations of Serum IgG Fucosylation as a Potential Additional New Diagnostic Marker in Advanced Endometriosis

doi: 10.2147/JIR.S341906

Figure Lengend Snippet: The Correlations Between Relative Reactivities of IgG Glycans with Fucose-Specific Lectins

Article Snippet: The method was based on the reactivity of IgG sugar moieties with four fucose-specific biotinylated lectins (Vector Laboratories Inc., Burlingame, CA, USA): 1) Aleuria aurantia lectin (AAL, recognizing fucose α1,6-linked to the N-acetylglucosamine core of N-glycans and with lower affinity fucoses α1,2-, α1,3- and α1,4-linked of the outer arms, 2) Lens culinaris agglutinin (LCA, recognizing sequences containing fucosylated tri-mannose N-glycan core sites), 3) Lotus tetragonolobus agglutinin (LTA, specifically reacting with fucose α1,3-linked to GlcNAc, characteristic for Lewis x oligosaccharide structures, however, it can also slightly react with fucose typical for Lewis a and Lewis y structures.

Techniques:

Journal: Journal of Inflammation Research

Article Title: The Alterations of Serum IgG Fucosylation as a Potential Additional New Diagnostic Marker in Advanced Endometriosis

doi: 10.2147/JIR.S341906

Figure Lengend Snippet: The correlations between relative reactivities of s-IgG and i–IgG glycans with fucose-specific lectins ( A – D ). For lectins’ specificity see Materials and methods section. A two-tailed p-value of less than 0.05 was considered as significant.

Article Snippet: The method was based on the reactivity of IgG sugar moieties with four fucose-specific biotinylated lectins (Vector Laboratories Inc., Burlingame, CA, USA): 1) Aleuria aurantia lectin (AAL, recognizing fucose α1,6-linked to the N-acetylglucosamine core of N-glycans and with lower affinity fucoses α1,2-, α1,3- and α1,4-linked of the outer arms, 2) Lens culinaris agglutinin (LCA, recognizing sequences containing fucosylated tri-mannose N-glycan core sites), 3) Lotus tetragonolobus agglutinin (LTA, specifically reacting with fucose α1,3-linked to GlcNAc, characteristic for Lewis x oligosaccharide structures, however, it can also slightly react with fucose typical for Lewis a and Lewis y structures.

Techniques: Two Tailed Test

Journal: Journal of Inflammation Research

Article Title: The Alterations of Serum IgG Fucosylation as a Potential Additional New Diagnostic Marker in Advanced Endometriosis

doi: 10.2147/JIR.S341906

Figure Lengend Snippet: Receiver operating characteristic curve analysis of serum native IgG - s-IgG (s) and isolated serum IgG - i–IgG (i) relative reactivities with AAL ( Aleuria aurantia lectin), LCA ( Lens culinaris agglutinin), LTA ( Lotus tetragonolobus agglutinin) and UEA ( Ulex europaeus agglutinin) was done for women with endometriosis and healthy subjects ( A – H ). For lectins’ specificity see Materials and methods section.

Article Snippet: The method was based on the reactivity of IgG sugar moieties with four fucose-specific biotinylated lectins (Vector Laboratories Inc., Burlingame, CA, USA): 1) Aleuria aurantia lectin (AAL, recognizing fucose α1,6-linked to the N-acetylglucosamine core of N-glycans and with lower affinity fucoses α1,2-, α1,3- and α1,4-linked of the outer arms, 2) Lens culinaris agglutinin (LCA, recognizing sequences containing fucosylated tri-mannose N-glycan core sites), 3) Lotus tetragonolobus agglutinin (LTA, specifically reacting with fucose α1,3-linked to GlcNAc, characteristic for Lewis x oligosaccharide structures, however, it can also slightly react with fucose typical for Lewis a and Lewis y structures.

Techniques: Isolation

Journal: Cells

Article Title: Fucosylated Proteome Profiling Identifies a Fucosylated, Non-Ribosomal, Stress-Responsive Species of Ribosomal Protein S3

doi: 10.3390/cells10061310

Figure Lengend Snippet: UEAI recognizes intracellular proteins. ( A ) WM793 cells were fixed and permeabilized with Triton X-100 or left unpermeabilized. Cells were subsequently stained with DAPI nuclear stain, FITC-AAL, FITC-UEAI, or no lectin as a control. Cell were visualized by IF for qualitative lectin staining (left panel). For quantification, WM793 and A375 cells were stained as above and analyzed by flow cytometry (right panel; MFI: Mean Fluorescence Intensity (of UEA1 per cell)). * p = 0.006 ( B ) Cells transduced and selected with lentivirus containing Cas9/no guide (ng) or Cas9/sgSLC35C1 (gC1) were grown in normal growth media and harvested for protein lysate. Lysates were probed with AAL and UEAI lectin to determine global levels of Golgi-dependent fucosylation. ( C ) WM793 cells were cultured for three days in 2-fluorofucose (2-FF, FUTi, 250 µM), L-fucose (L-fuc, 250 µM), or control. Cells were harvested for protein lysate and blotted for global AAL and UEAI levels.

Article Snippet: UEAI, AAL, UEAI-488, AAL-488, UEAI-biotin, AAL-biotin were purchased from Vector Laboratories (Burlingame, CA, USA).

Techniques: Staining, Flow Cytometry, Fluorescence, Cell Culture

Journal: Cells

Article Title: Fucosylated Proteome Profiling Identifies a Fucosylated, Non-Ribosomal, Stress-Responsive Species of Ribosomal Protein S3

doi: 10.3390/cells10061310

Figure Lengend Snippet: RPS3 is fucosylated in vitro and in vivo. ( A ) RPS3 is recognized by UEAI lectin by agarose-bound lectin pull-down (PD). Cell lines IPC298, MelJuso, and SK-MEL-119 were cultured in growth media and harvested for protein lysate. Lysates were incubated with bead (agarose bead control), AAL (AAL-agarose), or UEAI (UEAI-agarose) to bind fucosylated proteins. UEAI-recognized RPS3 in all three cell lines. ( B ) Liver, muscle, brain, and skin samples were harvested from a C57BL/6 mouse and lysed using a Dounce homogenizer. Lysates were subjected to UEAI PD to isolate UEAI-recognized proteins. RPS3 was recognized by UEAI in all tissue types. LE: long exposure. ( C ) Cells transduced and selected with lentivirus containing Cas9/no guide (ng) or Cas9/sgSLC35C1 (gC1) were grown in normal growth media and harvested for protein lysate ( B). Lysate were subjected to UEAI PD to isolate UEAI-recognized proteins. Knockout of the Golgi fucose transporter SLC35C1 did not affect the ability of UEAI to recognize RPS3. ( D ) Cells expressing myc-DDK (FLAG)-tagged RPS3 (RPS3-mDDK) were harvested for FLAG IP and Western blot. The membrane was probed with mouse anti-FLAG/anti-mouse-680 and UEAI-biotin/strep-800 to assess direct recognition of RPS3 by UEAI (signal overlap by LI-COR imaging). UEAI recognizes RPS3. ( E ) Cells were grown in normal growth media and harvested for UEAI PD. The lectin PD was supplemented with either no sugar (control), L-fucose (Fuc), glucose (Glc), rhamnose (Rham), or galactose (Gal) at 1 M final concentration. Analysis by Western blot showed that fucose and rhamnose interfere with UEAI lectin recognition of RPS3, whereas glucose and galactose did not affect lectin binding. ( F ) Cells grown in the presence of labeled fucose were analyzed to assess labeling of RPS3. Labeled lysates or unlabeled lysates (control) were subjected to neutravidin pull-down (Neu-PD) to isolate proteins that integrated the labeled fucose. RPS3 did integrate the labeled fucose. β-actin, a non-fucosylated protein, did not show any integration of the labeled fucose (negative control).

Article Snippet: UEAI, AAL, UEAI-488, AAL-488, UEAI-biotin, AAL-biotin were purchased from Vector Laboratories (Burlingame, CA, USA).

Techniques: In Vitro, In Vivo, Cell Culture, Incubation, Knock-Out, Expressing, Western Blot, Imaging, Concentration Assay, Binding Assay, Labeling, Negative Control