Analysis of N-linked oligosaccharide chains of glycoproteins on nitrocellulose sheets using lectin-peroxidase reagents

Kijimoto-Ochiai, Shigeko; Katagiri, Yohko U.; Ochiai, Hiroshi

doi:10.1016/0003-2697(85)90031-4

Cited by 134 publications

(32 citation statements)

References 25 publications

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“…10,11) Proteins in the gel were stained with Coomassie Brilliant Blue (CBB) or transferred to a nitrocellulose membrane. The membrane was then stained with horseradish peroxidase (HRP)-conjugated anti-GST antibody (Amersham) to detect GST fusion proteins.…”

Section: Expression and Puriˆcation Of Fusion Proteinmentioning

confidence: 99%

Carbohydrate Binding Specificity of the Recombinant Chitin-binding Domain of Human Macrophage Chitinase

Ujita

Sakai

Hamazaki

et al. 2003

Bioscience, Biotechnology, and Biochemistry

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Section: Expression and Puriˆcation Of Fusion Proteinmentioning

confidence: 99%

Carbohydrate Binding Specificity of the Recombinant Chitin-binding Domain of Human Macrophage Chitinase

Ujita

Sakai

Hamazaki

et al. 2003

Bioscience, Biotechnology, and Biochemistry

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“…Lectin staining was performed as described by Kijimoto-Ochiai et al [23,24] with some modifications [25]. In brief, the blotted nitrocellulose membrane was washed twice in Tris-Tween-saline (pH 7.4) composed of 10 mM Tris-HCl, 0.05% Tween 20 and 0.15 M NaCl for 10 min each, and blocked with 5% bovine serum albumin (BSA, Fraction V, Seikagakukogyo, Tokyo, Japan) in Tris-Tween-saline for 1 h. Each of the lectin-peroxidase reagents (final concentration: 0.5 µg protein/ml) was diluted with ice-cold Tris-Tweensaline with (Con A and LCA) or without 1 mM CaCl 2 , 1 mM MgCl 2 and 1 mM MnCl 2 (RCA I and PNA).…”

Section: Lectin Blottingmentioning

confidence: 99%

Lectin-Binding Characteristics of Extracts from Epididymal Boar Spermatozoa.

Harayama

Watanabe²,

Masuda³

et al. 1998

Journal of Reproduction and Development

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Abstract. Glycosylated components of boar spermatozoa and epididymal plasma from the central caput, distal corpus and distal cauda epididymides were compared by electrophoretic and lectin blotting techniques. The four lectin reagents used in this study were Concanavalin A (Con A), Lens Culinaris Agglutinin (LCA), Ricinus Communis Agglutinin I (RCA I) and Peanut Agglutinin (PNA), which were labeled with peroxidase. The affinity of Con A and of LCA increased for three bands and one smear (A: >86.8 kDa, B: 80 kDa, C: 65-75 kDa and D: 45 kDa) and for two bands and one smear (A, B and C) of extracts from spermatozoa, respectively, as they passed through the epididymis. RCA I apparently recognized one band and one smear (G: >86.8 kDa and H: 46-58 kDa) of extracts from the distal cauda spermatozoa, though it hardly or faintly reacted with those from the central caput and distal corpus spermatozoa. PNA showed a similar affinity for band "G" to RCA I. Moreover, bands and smears with similar mobility (A, B, C, D, G and H) were also detected in epididymal plasma by lectin blotting techniques. On the other hand, several sperm components were detected more visibly with Con A (E: 37-41 kDa and F: 32-37 kDa), RCA I (I: 41 kDa) and PNA (I: 41 kDa and J: 47-51 kDa) in sperm extracts from the distal cauda than in those from the central caput, though no corresponding components were observed in epididymal plasma. These epididymal maturation-dependent modifications related to glycosylation in boar spermatozoa are discussed.

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“…Proteins on a gel were transferred to nitrocellulose sheets in 25 mM Tris, 192 mM glycine, 20% methanol (v/v) [26] at 250 mA for 1 h and 300 mA for 2 h. The nitrocellulose sheet with blotted proteins was washed twice in 30 ml10 mM Tris/HCl (pH 7.9, 0.05% Tween 20, 0.15 M NaCl (buffer A) containing 2% bovine serum albumin, then twice with buffer A.…”

Section: Sds-page and Western Blottingmentioning

confidence: 99%

“…The treated nitrocellulose sheet was washed four times in buffer A, then reacted with 3,3'-diaminobenzidine [26]. After staining, the nitrocellulose sheet was washed several times with water and dried.…”

Section: Interaction Of Cta With Hrp-lectimsmentioning

confidence: 99%

Characterization of the carbohydrate moiety of Clerodendron trichotomum lectins

Kitagaki-Ogawa

Matsumoto

Seno

et al. 1986

European Journal of Biochemistry

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Lectins were isolated from fruits and leaves of Clerodendron trichotomum by affinity chromatograpy on lactamyl-Sepharose. The purified lectins (C. trichotomum agglutinin : CTA) were homogeneous on SDS/polyacrylamide gel electrophoresis, and the carbohydrate moiety was characterized by physicochemical and immunochemical methods.The asparagine-linked oligosaccharides were released by treatment with N-oligosaccharide glycopeptidase (almond, EC 3.5.1.52) of peptic glycopeptides obtained from fruit CTA, and separated by gel filtration and thin-layer chromatography. The structure of the predominant oligosaccharide was determined as Xylpl -, 2 (Manal -+ 6)(Man a1 -+ 3)Man/31 + 4GlcNAcBl -+ 4(Fucal -, 3)GlcNAc by high-performance liquid chromatography, sugar analysis and 'H-NMR spectroscopy.The reactivity of the carbohydrate moiety of CTA toward various lectins was studied. Fruit and leaf CTAs were applied to polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets and detected with horseradishperoxidase-conjugated lectins.

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Analysis of N-linked oligosaccharide chains of glycoproteins on nitrocellulose sheets using lectin-peroxidase reagents

Cited by 134 publications

References 25 publications

Carbohydrate Binding Specificity of the Recombinant Chitin-binding Domain of Human Macrophage Chitinase

Carbohydrate Binding Specificity of the Recombinant Chitin-binding Domain of Human Macrophage Chitinase

Lectin-Binding Characteristics of Extracts from Epididymal Boar Spermatozoa.

Characterization of the carbohydrate moiety of Clerodendron trichotomum lectins

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