α2,3 and α2,6 N-linked Sias allow for efficient interaction of NDV with target cells [27 (link)]. SNA binds preferentially to Sias attached to terminal galactose through the α2,6 linkage and, to a lesser degree, α2,3 linkage. MAL1 binds to Gal (β-1,4) GlcNAc but tolerates the substitution of N-acetyllactosamine with Sia at the 3 position of galactose. After harvesting, cells were fixed with 4% PFA at room temperature for 30 min. Lectin staining was performed with fluorescein-labeled Maackia amurensis lectin I (MAL1; Vector Laboratories, San Mateo, CA, USA) and Sambucus nigra lectin (SNA; Vector Laboratories, CA, USA) according to the manufacturer’s instructions. MAL1 binds to Gal (β-1,4) GlcNAc but tolerates the substitution of N-acetyllactosamine with Sia at the 3 position of galactose, while SNA binds to α2,6-linked Sia. The respective lectins were added to the cell cultures, and the samples were incubated at 37 °C for 30 min and then rinsed three times with PBS. The binding of the labeled lectins to cells was detected using flow cytometry.
Maackia amurensis lectin 1 mal 1
Manufactured by Vector Laboratories
Sourced in United States
Maackia amurensis lectin I (MAL-I) is a carbohydrate-binding protein derived from the bark of the Amur maackia tree. It has affinity for sialic acid-containing glycans. MAL-I can be utilized for the detection and purification of sialic acid-bearing molecules.
Automatically generated - may contain errors
Lab products found in correlation
Sambucus nigra lectin sna, by Vector Laboratories (3 mentions)
Sambucus nigra agglutinin, by Vector Laboratories (1 mentions)
Accuri flow cytometer, by BD (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Vitamin c, by Merck Group (1 mentions)
2 7 dichlorofluorescin diacetate dcfh da, by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Anhydrous dimethyl sulfoxide dmso, by Merck Group (1 mentions)
Rosup, by Beyotime (1 mentions)
Anatase nano tio2, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Erythrina cristagalli lectin eca, by Vector Laboratories (1 mentions)
Octet red384, by Molecular Devices (1 mentions)
Phosphate buffered saline (pbs), by Lonza (1 mentions)
Helix pomatia agglutinin hpa, by Merck Group (1 mentions)
Oseltamivir, by Merck Group (1 mentions)
Alexa fluor 635 streptavidin, by Thermo Fisher Scientific (1 mentions)
Erythrina cristagalli agglutinin, by Vector Laboratories (1 mentions)
7 protocols using maackia amurensis lectin 1 mal 1
1
Efficient NDV Cell Interaction via Sialic Acids
2
Lectin-Based Organoid Cell Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In brief, organoids were dissociated using TrypLE, centrifuged at 500 x g for 5 min, and resuspended in FACS buffer (PBS with 2% FBS). The fluorescein-conjugated lectins, Sambucus nigra agglutinin (SNA) (Vector Laboratories, FL-1301, USA) and Maackia Amurensis Lectin I (MAL 1) (Vector Laboratories, FL-1311, USA) were then diluted in a 1:500 and 1:100 ratio, respectively, in FACS buffer. The dissociated cells were then mixed with the lectins and incubated for 30 min in the dark on ice. The cells were washed twice with 1 ml FACS buffer, centrifuged at 500 x g for 5 min and resuspended in 500 µl FACS buffer, before the samples were run on an Accuri Flow Cytometer (BD Biosciences) and data were analyzed by Cytobank (Santa Clara, CA, USA).
3
Nano-TiO2 Characterization and Cell Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nano-TiO2, an anatase-rutile mixture of nano-TiO2 P25 (Degussa Company, Essen, Germany), anatase nano-TiO2 (Sigma Company, St. Louis, MO, USA) and rutile nano-TiO2 (Macklin, Shanghai, China) were obtained from commercial sources. UV light was generated by an ultraviolet lamp (ZF-5, 365 nm, 8 W, 0.6 mW/cm2, Shanghai Huxi Instrument, Shanghai, China).
Fetal bovine serum (FBS), Dulbecco’s modified Eagle medium (DMEM), phosphate-buffered saline (PBS, pH 7.4), penicillin, streptomycin, and trypsin-EDTA were purchased from Gibco (Invitrogen, Carlsbad, CA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma (St. Louis, MO, USA). Sambucus nigra lectin (SNA) labeled with fluoresceinIsothiocyanate (FITC) FITC and Maackia amurensis lectin I (MAL-I) were purchased from Vector labs (Burlingame, CA, USA).
Anhydrous dimethyl sulfoxide (DMSO) and vitamin C (VC) were purchased from Sigma Aldrich (St. Louis, MO, USA). ROSup was purchased from Beyotime (Shanghai, China).
Fetal bovine serum (FBS), Dulbecco’s modified Eagle medium (DMEM), phosphate-buffered saline (PBS, pH 7.4), penicillin, streptomycin, and trypsin-EDTA were purchased from Gibco (Invitrogen, Carlsbad, CA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma (St. Louis, MO, USA). Sambucus nigra lectin (SNA) labeled with fluoresceinIsothiocyanate (FITC) FITC and Maackia amurensis lectin I (MAL-I) were purchased from Vector labs (Burlingame, CA, USA).
Anhydrous dimethyl sulfoxide (DMSO) and vitamin C (VC) were purchased from Sigma Aldrich (St. Louis, MO, USA). ROSup was purchased from Beyotime (Shanghai, China).
4
Real-time virus binding analysis by BLI
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Real-time virus binding was studied by BLI analysis using an Octet RED384 (Fortebio). All experiments were carried out at 30 °C in Dulbecco’s PBS with calcium and magnesium (PBS+/+) (Lonza) as standard assay buffer. BLI protocols are described stepwise in detail in ref. 46 (link). In short, SA biosensors were loaded with biotinylated receptors. These were either biotinylated synthetic glycans [2-6Sia-(LN)3, Siaα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc; 2-6Sia-(LN)2, Siaα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1 or the Siaα2-3 versions 2-3Sia-(LN)3 or 2-3Sia-(LN)2 of these] or recombinant glycoprotein LAMP1 (2-6Sia-LAMP1 or 2-3Sia-LAMP 1). Receptors were loaded to saturating levels and real-time virus association was examined for 900 s in the presence of 10 µM oseltamivir carboxylate (OC; Roche) to block NA activity. Absolute initial virus-binding rates were calculated and plotted (nm/109 virus particles). LAMP1 sialylation levels were analyzed by lectin binding assays [(Maackia amurensis lectin I (MAL I, Vector Labs) binds to α2-3Sia-Galβ1-4GlcNAc; Sambucus nigra lectin (SNA, Vector Labs) binds preferentially to α2- 6Sia-Galβ1-4GlcNAc; and Erythrina cristagalli lectin (ECA, Vector Labs) binds to terminal LacNAc (Galβ1-4GlcNAc)].
5
Biotinylated Lectin Binding Assay
6
Virus Isolation and Antibody Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Virus isolates were produced as described above. Oseltamivir was purchased from Sigma Aldrich [Cat# SML1606]. CR8020 A/H3N2 stem antibody was kindly provided by Dr. Dirk Eggink and expressed following previously published procedures48 (link). Goat anti-human Alexa-647 [Cat# A21445] and streptavidin-AlexaFluor 635 [Cat# SA1011] antibodies were obtained from Thermo Fisher. Control lectins Erythrina cristagalli agglutinin (ECA) [Cat#B-1145], Sambuca nigra agglutinin (SNA) [Cat# B-1305], and Maackia amurensis lectin I (Mal-I) [Cat# B-1315] were purchased from Vector Labs.
7
PAMAM Dendrimer Glycoconjugation and Sialic Acid Validation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The generation 2.0 PAMAM dendrimer with a cystamine core (647829, Sigma Aldrich) was conjugated to three different glycans via reductive amination. 20 equivalents of LS-Tetrasaccharide d (LSTd, Elicityl) and D-(+)-Galactose (G0750 Sigma Aldrich) per dendrimer were dissolved in Dimethylsulphoxide (DMSO) and acetic acid (8:2). Per dendrimer, 160 equivalents 2-Methylpyridine borane complex (65421 Sigma Aldrich) was added to a total volume of 200 µL. The reaction was incubated at 65 °C for 2 h with frequent vortexing. The reaction products were purified over disposable PD10 desalting columns (GE17-0851-01 GE Healthcare) in 50 mM Ammonium Formate pH 4.4 (NH4HCO3).
The presence of α2-3 sialic acid was validated using an ELISA-type assay using Maackia Amurensis Lectin I (MAL-I) (Vector Laboratories, Peterborough, UK). Briefly, NUNC maxisorb plates (RosKilde) were coated overnight at 4 °C with 5 µM of the products. The wells were subsequently blocked for 2 h at room temperature with carbo-free blocking buffer (Vector, SP5040). Incubation with the biotinylated MAL-I and peroxidase-labelled streptavidin (Sigma-Aldrich) allowed spectrophotometric quantification of the binding with 3,3′,5,5′-tetramethylbenzidine (TMB, Sigma-Aldrich) at 450 nm on the iMarkTM Microplate Absorbance Reader (Bio-RAD).
The presence of α2-3 sialic acid was validated using an ELISA-type assay using Maackia Amurensis Lectin I (MAL-I) (Vector Laboratories, Peterborough, UK). Briefly, NUNC maxisorb plates (RosKilde) were coated overnight at 4 °C with 5 µM of the products. The wells were subsequently blocked for 2 h at room temperature with carbo-free blocking buffer (Vector, SP5040). Incubation with the biotinylated MAL-I and peroxidase-labelled streptavidin (Sigma-Aldrich) allowed spectrophotometric quantification of the binding with 3,3′,5,5′-tetramethylbenzidine (TMB, Sigma-Aldrich) at 450 nm on the iMarkTM Microplate Absorbance Reader (Bio-RAD).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!