Equal proteins extracted from cells were additionally separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore). Membranes were blocked for nonspecific binding with 3% BSA-TBST at room temperature for 1 h and then incubated with biotinylated Maackia amurensis lectin I (MAL-I; catalog no., B-1315; Vector Laboratories, Inc., Burlingame, CA, USA), biotinylated leucoagglutinin (PHA-L; catalog no., B-1115; Vector Laboratories, Inc.) at a dilution of 1:2,000 or a primary monoclonal mouse anti-human β-actin antibody (cat. no. KC-5A08; KangCheng Bio-tech, Shanghai, China) at a dilution of 1:2,000 at room temperature for 30 min. Subsequent to washing with 0.1% TBST for 10 min 3 times, membranes were incubated with streptavidin horseradish peroxidase (HRP) conjugate (Invitrogen; Thermo Fisher Scientific, Inc.) at a dilution of 1:10,000 or HRP-conjugated secondary antibody (cat. no. KC-MM-035; KangCheng Bio-tech) at a dilution of 1:10,000 at room temperature for another 30 min, and washed by 0.1% TBST for 10 min 3 times. The bands on the membranes were detected by using Amersham enhanced chemiluminescence (ECL) prime western blotting detection reagents (GE Healthcare). Anti-β-actin antibody was used as the protein loading control.
Biotinylated maackia amurensis lectin 1 mal 1
Manufactured by Vector Laboratories
Sourced in United States
Biotinylated Maackia amurensis lectin I (MAL-I) is a glycoprotein derived from the Amur Maackia tree. It functions as a specific binding agent for sialic acid-containing glycoconjugates.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin horseradish peroxidase hrp conjugate, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti flag, by Merck Group (1 mentions)
Neu a, by New England Biolabs (1 mentions)
Neu s, by New England Biolabs (1 mentions)
Biotinylated sambucus nigra agglutinin sna, by Vector Laboratories (1 mentions)
Fusion solo x, by Vilber (1 mentions)
Horseradish peroxidase conjugated goat anti mouse igg, by Bioss Antibodies (1 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
Cytoflex flow cytometry, by Beckman Coulter (1 mentions)
Streptavidin percp, by BD (1 mentions)
Kaluza analysis software version 2, by Beckman Coulter (1 mentions)
Cytofix cytoperm reagent, by BD (1 mentions)
Pe conjugated anti cd20, by BD (1 mentions)
Fitc conjugated anti np antibody, by Merck Group (1 mentions)
Perm wash, by BD (1 mentions)
3 protocols using biotinylated maackia amurensis lectin 1 mal 1
1
Immunoblotting Analysis of Glycoproteins
2
Glycan Profiling of Nanoparticles
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Nanoparticles were incubated with neuraminidase A (Neu A, New England biolabs, Inc., Beverly, MA, USA), neuraminidase S (Neu S, New England biolabs, Inc.), or PNGase F (New England biolabs, Inc.) at 37 °C for 18 h. Proteins were transferred to a polyvinylidene fluoride membrane (Thermo Fisher Scientific, Waltham, MA, USA). After blocking with 5% (w/v) bovine serum albumin in Tris-buffered saline with 0.05% TWEEN 20 (TBST) for 1 h at 25 °C, membranes were probed with monoclonal mouse anti-flag (1:1000, Sigma-Aldrich, St. Louis, MO, USA) antibodies, biotinylated Sambucus nigra agglutinin (SNA, Vector Laboratories, Burlingame, CA, USA), or biotinylated Maackia amurensis lectin-I (MAL-I, Vector Laboratories, Burlingame, CA, USA) as a primary staining, and then with horseradish peroxidase-conjugated goat anti-mouse IgG (Bioss Antibodies, Woburn, MA, USA) and streptavidin at 1:5000 dilution in TBST. Membranes were developed using ECL substrate (Thermo Fisher Scientific, Waltham, MA, USA) and signals were detected with Fusion Solo X (Vilber, Paris, France).
3
Flow Cytometric Analysis of Virus-Infected Cells
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Cells were washed with 0.1% BSA in PBS, followed by specific surface staining with 1:50 APC-conjugated anti-CD14 (immunotools, Friesoythe, Germany) and 1:20 PE-conjugated anti-CD20 (BD biosciences, CA, USA) at 4°C for 30 min. Cells were then washed and fixed with 4% paraformaldehyde at 4°C for 30 min. Subsequently, cells were stained for surface α2,3 SA with 1:100 biotinylated Maackia Amurensis Lectin I (MAL I) (Vector Laboratories, CA, USA) at 4°C for 30 min. After washing, cells were incubated with 1:200 Per-CP Streptavidin (BD Biosciences, CA, USA). For intracellular staining of viral NP, cells were permeabilized in Cytofix/Cytoperm reagent (BD Biosciences, CA, USA) at 4°C for 10 min, then washed with Perm/wash (BD Biosciences, CA, USA), incubated with 1:500 FITC-conjugated anti-NP antibody (Millipore, USA) at 4°C for 30 min, washed and suspended in 2% paraformaldehyde. To detect α2,3 SA expression for single stain, cells were stained with 1:100 FITC-conjugated MAL I (Vector Laboratories, CA, USA). Stained cells were detected using a CytoFLEX flow cytometry (Beckman Coulter, USA), and the data were analyzed using Kaluza Analysis Software—Version 2.0. (Beckman Coulter, USA). A total of 10,000 cells were acquired for each flow cytometry analysis.
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