Biotinylated lectins (Vector Laboratories), Maackia amurensis Lectin II (MAL-II), Sambucas nigra Agglutinin (SNA), Aleuria aurantia (AAL) and Ulex europaeus (U.Euros) were used in conjunction with streptavidin PE-Cy7 or phycoerythrin (PE) for ow cytometric analysis. The lectins were titrated to minimise agglutination, as indicated by abnormally high forward versus side scatter (FCS) and high PE-Cy7 or PE intensity. Optimal ow cytometry concentrations for the lectins were MAL-II: 67 ng/ml; SNA: 57ng/ml; AAL: 200ng/ml; and U.Euros: 100ng/ml. Biotinylated lectins were applied, followed by streptavidin to puri ed erythrocytes, post-blocking with Carbo-Free-Block (Vector, SP5040) at room temperature for 30 minutes each before subjecting to ow cytometry (Dodge, Mitchell & Hanahan, 1963) .
Biotinylated lectin
Manufactured by Vector Laboratories
Sourced in United States
Biotinylated lectins are a class of glycan-binding proteins derived from plants, animals, and microorganisms. They are covalently labeled with biotin, a small molecule that can be used to attach the lectin to various substrates or detection systems. Biotinylated lectins are widely used in biochemical and cell biology applications to identify and study the distribution of specific carbohydrate structures on the surfaces of cells and in biological samples.
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Lab products found in correlation
Horseradish peroxidase avidin d, by Vector Laboratories (2 mentions)
Streptavidin alexa 488, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Sa3800, by Sony (2 mentions)
Hrp conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Europium conjugated streptavidin, by PerkinElmer (1 mentions)
Microlon high binding, by Greiner (1 mentions)
Carbohydrate free blocking solution, by Vector Laboratories (1 mentions)
Streptavidin horseradish peroxidase, by Vector Laboratories (1 mentions)
Blocking buffer, by LI COR (1 mentions)
Immobilon fl pdvf membranes, by Merck Group (1 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
Streptavidin poly hrp, by Thermo Fisher Scientific (1 mentions)
Chemiluminescence detection reagent, by GE Healthcare (1 mentions)
Amaranthus caudatus lectin, by Vector Laboratories (1 mentions)
Sa3800 spectral analyzer, by Sony (1 mentions)
Alexa flour 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 or 647, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated rabbit anti mouse immunoglobulins, by Agilent Technologies (1 mentions)
43 protocols using biotinylated lectin
1
Lectin-Based Flow Cytometry Analysis
2
Lectin Histochemistry for Glycan Profiling
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A battery of seven biotinylated lectins (Vector Laboratories, Inc. Burlingame, CA) was used to identify specific sugar residues (Table 1). The paraffin sections were mounted on slides treated with poly-L-lysine (Sigma Diagnostics, St Louis, MO), deparaffinated with xylol and incubated in a 0.3% H 2 O 2 solution in methanol for 30 min at room temperature to inhibit the endogenous peroxidase activity.
Then, the sections were hydrated, washed with 0.01 mol L 21 , pH 7.6 saline phosphate buffer (SPB) and incubated with a bovine serum albumin in SPB for 20 min to block specific bindings. After rinsing, sections were incubated with each of the biotinylated lectins for 30 min at room temperature and treated with avidine-biotineperoxidase complex (ABC) during 45 min (Vector Laboratories, Inc).
The peroxidase was activated through 4-10 min incubation with a Tris-HCl 0.05 mol L 21 , pH 7.6 tamponed solution containing 0.02% diaminobenzine (DAB; Dako, Carpinteria, CA, EE.UU.), and 0.05% H 2 O 2 .
All lectins were employed in a 30 mg ml -1 in SPB, except for PNA that was applied at concentration 10 mg ml -1 .
The results were semi-quantitatively evaluated, using the same scale as the one used for the histochemical study. Two types of controls were made: (1) the lectin solution was replaced by SPB and (2) lectins were preincubated at ambient temperature for 1 hr in presence of adequate haptens.
Then, the sections were hydrated, washed with 0.01 mol L 21 , pH 7.6 saline phosphate buffer (SPB) and incubated with a bovine serum albumin in SPB for 20 min to block specific bindings. After rinsing, sections were incubated with each of the biotinylated lectins for 30 min at room temperature and treated with avidine-biotineperoxidase complex (ABC) during 45 min (Vector Laboratories, Inc).
The peroxidase was activated through 4-10 min incubation with a Tris-HCl 0.05 mol L 21 , pH 7.6 tamponed solution containing 0.02% diaminobenzine (DAB; Dako, Carpinteria, CA, EE.UU.), and 0.05% H 2 O 2 .
All lectins were employed in a 30 mg ml -1 in SPB, except for PNA that was applied at concentration 10 mg ml -1 .
The results were semi-quantitatively evaluated, using the same scale as the one used for the histochemical study. Two types of controls were made: (1) the lectin solution was replaced by SPB and (2) lectins were preincubated at ambient temperature for 1 hr in presence of adequate haptens.
3
Quantification of Glycan Modifications
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Titers of fucosylated or sialylated AGPs or of high-mannose-type glycan were determined as previously, with modifications [23 (link)]. The samples were coated in the same manner as for ELISA and after washing with Tris- buffered saline (TBS) containing 0.1% Tween 20 (TBST), the wells were blocked with TBS containing 1% Tween 20 at room temperature for 2 h to avoid nonspecific reactions with lectins. Biotinylated lectins were purchased from Vector Laboratories (USA). Aleuria Aurantia Lectin (AAL) and jacalin were used to detect fucosylation and mannosylation, respectively, and Maackia Amurensis Lectin 2 (MAA) was used to detect sialylation. All lectins were diluted to 1 μg/ml in TBST and reacted with the AGP-coated wells at room temperature for 1 h, and bound lectins were detected with HRP-conjugated streptavidin (Thermo Fisher Scientific, 1:10,000 dilution). The plates were washed three to five times with TBST between reactions. Color reactions were developed as described above. End-point titers were set at an OD of 2 × the OD of the control well.
4
Lectin ELISA for Purified IgG
5
Immunohistochemical Analysis of Cholangiocytes
6
Lectin-Binding Assay for Fc Proteins
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Black 384-well Maxisorp Nunc microtiter plates (Fisher Scientific, Waltham, MA) were coated with recombinant Fc proteins (rFc) or IVIG in phosphate-buffered saline (PBS) (1 µg/mL; 50 µL/well) for 30 min at room temperature (RT). The plates were washed to remove unbound proteins and blocked with PBSAT 1% bovine serum albumin (BSA) and 0.5% Tween-20 in PBS for 20 min. Biotinylated lectins (2–10 µg/mL; Vector Labs, Burlingame, CA) were precomplexed with neutravidin-HRP (Pierce, Rockville, IL) on ice for 20 min and 50 µL of the lectin-neutravidin-HRP complex added to each well; plates were incubated at room temperature for 2 h and washed six times with PBSAT to remove unbound or weakly bound lectins. HRP activity was measured using Amplex Red Peroxidase Assay Kit (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. Appropriate negative and positive controls were included, and the tests were run in quadruplicate.
7
Glycan Profile Analysis of A2780 and A2780-cp Cells
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The total proteins isolated from the A2780 cells and A2780-cp cells were analyzed via SDS-PAGE and lectin blotting. Briefly, the samples were separated via 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking with TBST (150 mM NaCl, 10 mM Tris–HCl, and 0.05% v/v Tween 20, pH 7.5) containing 5% bovine serum albumin for 1 h at room temperature, the PVDF membranes were incubated with the biotinylated lectins (Vector Laboratories, Burlingame, CA, USA) from Lens culinaris (LCA), Canavalia ensiformis (ConA), Lycopersicon esculentum (LEL) and Sambucus nigra (SNA) for 2 h at room temperature. The membranes were subsequently incubated with horseradish peroxidase streptavidin (Vector Laboratories) for 30 min and detected with an ECL assay kit.
8
Western Blot Protocol for Protein Detection
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Whole cell lysates were collected in lysis buffer containing 1x protease inhibitor cocktail. Protein concentration was measured by the BCA protein assay (Thermo Scientific, Rockford, IL), separated by electrophoresis through 4–12% or 7.5% SDS-PAGE and then transferred to Immobilon-FL PDVF membranes (Millipore, Billerica, MA). Membranes were blocked in LiCor blocking buffer (LiCor, Lincoln, NE) diluted with PBS (1:1), then incubated with primary antibodies overnight at 4°C. Following extensive washing, membranes were incubated with species-specific IRDye700 or 800-conjugated secondary antibodies (1:15,000) for 1 h at room temperature, and visualized with a LiCor Odyssey infrared imager (LiCor, Lincoln, NE). Consistent protein loading was determined by reprobing membranes with anti-actin or anti-GAPDH antibodies. To probe with lectins, the protein blot was blocked with protein-free blocking buffer (PFB, Thermo Scientific, Rockford, IL) at room temperature for 1 h, and then probed overnight at 4°C with 1 μg/ml of biotinylated lectins (Vector Laboratories, Burlingame, CA) in lectin-binding buffer (50 mM Tris, pH 7.5, 0.15 M NaCl, 0.1 mM CaCl2). The filters were then washed with TBST (TBS with 0.05% Tween-20), incubated with IRDye 800-conjugated streptavidin in PFB for 1 h and then washed with TBST. The blots were imaged on the LiCor Odyssey system.
9
Lectin binding assay for rFVIII
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rFVIII products were adsorbed to Maxisorp microtitre plates at 1 μg/mL overnight at 4°C. All products saturated binding at this concentration (Online Supplementary Figure S1). Plates were blocked with 1% BSA in phosphate buffered saline (PBS) + 0.01% Tween-20 for 1 hr, and subsequently incubated with biotinylated lectins (Vector Laboratories, Burlingame, CA, USA) for 30 min. Detection was facilitated using streptavidin-poly-HRP (ThermoFisher Scientific, Waltham, MA, USA) and developed for 5 min. Statistical analysis was performed using the Student t test.
10
Lectin Blot Analysis of Hp Glycosylation
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Purified Hp of 60 μl of sera (pooled from 20 patients, 3 μl per patient) from LC and HCC, respectively, was analyzed by lectin blot according to the previous description (Shu et al., 2011 (link)). Five biotinylated lectins (Vector Laboratories, Burlingame, CA) including Amaranthus caudatus lectin (ACA, 1 μg/ml), Griffonia simplicifolia lectin I (GSI-L, 1 μg/ml), Jacalin (JAC, 1 μg/ml), Vicia villosa lectin (VVA, 1 μg/ml), and Wisteria floribunda lectin (WFA, 2 μg/ml) were incubated for 45 min, respectively. After washing and incubating with horseradish peroxidase Avidin D (Vector Laboratories, Burlingame, CA), the bands were developed using chemiluminescence detection reagents (GE Healthcare, Piscataway, NJ).
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