Biotinylated aal

Manufactured by Vector Laboratories

Sourced in United States

Biotinylated AAL is a reagent produced by Vector Laboratories. It is a lectin derived from the seeds of the Wisteria floribunda plant that has been conjugated with biotin.

Automatically generated - may contain errors

Lab products found in correlation

Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions) Multiscreen filter plate, by Merck Group (1 mentions) Amine coupling kit, by Bio-Rad (1 mentions) Cytokine assay kits, by Bio-Rad (1 mentions) Imagequant las 4000 imager, by GE Healthcare (1 mentions) Mouse anti gfp, by Roche (1 mentions) Extravidin hrp, by Merck Group (1 mentions) Pha l, by Vector Laboratories (1 mentions) Wheat germ agglutinin (wga), by Vector Laboratories (1 mentions) Mal 2, by Vector Laboratories (1 mentions) Chemiscope5600, by Clinx (1 mentions) Hrp labeled streptavidin, by Sangon (1 mentions) Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions) Uea 1, by Vector Laboratories (1 mentions) Neuraminidase a, by New England Biolabs (1 mentions) Neuraminidase s, by New England Biolabs (1 mentions) Mal 1, by Vector Laboratories (1 mentions) Pngase f, by New England Biolabs (1 mentions) Ecl western blotting detection reagent, by Thermo Fisher Scientific (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions)

7 protocols using biotinylated aal

Multiplexed Lectin-based Protein Detection

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Agarose-bound Aleuria Aurantia Lectin (AAL) beads were purchased from Vector Labs (Burlingame, CA). Multiscreen filter plates were from Millipore (Billerica, MA). Bio-Plex Pro^TM magnetic COOH beads, amine coupling kits, and cytokine assay kits were purchased from Bio-Rad Laboratories (Hercules, CA). Biotinylated AAL was purchased from Vector Labs (Burlingame, CA). Biotinylated detection antibody was prepared with Thermo Scientific (Rockford, IL) EZ-link Sulfo-NHS-Biotin (Catalog #21326).
Human recombinant PSA (Catalog #PO725), human PSA mouse monoclonal antibody (Catalog #MP077-BP001) for capture, and biotinylated mouse monoclonal antibody (Catalog #MP007-AP002S) for detection were purchased from Scripps Laboratories (San Diego, CA). Mouse myeloma cell line NS0-derived human recombinant TIMP1 (Catalog #970-TM-010), human TIMP1 mouse monoclonal IgG2B antibody (Catalog #MAB970, clone #63515) for capture, and biotinylated human TIMP1 goat polyclonal IgG antibody (Catalog # BAF970) for detection were purchased from R&D Systems (Minneapolis, MN). Chinese Hamster ovary cell line CHO-derived human recombinant tPA protein (Catalog #ab92637), human tPA mouse monoclonal antibody (Catalog # ab82249) for capture, and biotinylated human tPA rabbit polyclonal IgG antibody (Catalog #ab28208) for detection were purchased from Abcam (Cambridge, MA).

Li Q.K., Chen L., Ao M.H., Chiu J.H., Zhang Z., Zhang H, & Chan D.W. (2015). Serum Fucosylated Prostate-specific Antigen (PSA) Improves the Differentiation of Aggressive from Non-aggressive Prostate Cancers. Theranostics, 5(3), 267-276.

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Tachyzoite Protein Detection and Analysis

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About 5 × 10⁶ cells equivalent/lane T. gondii tachyzoites were loaded on 8–16% TGX gels (Life Technologies). After SDS-PAGE separation, proteins were blotted on PVDF, and then the membranes were blocked in 50 mM TrisHCl, 0.15 M NaCl, 0.25% BSA, 0.05% NP-40 pH 7.4. β-Elimination on blot was performed by incubating the membrane 16 h in 55 mM NaOH at 40 °C, under rotation (58 (link)), before blocking the membrane. Both primary and secondary antibodies were diluted in blocking buffer as follows: mouse MAb anti-cMYC 9E10 0.4 μg/ml, mouse MAb anti-α-tubulin 12G10 1:1000 (DHSB), biotinylated-AAL (Vector Labs) 2 μg/ml, mouse anti-GFP (Roche) 1:2000, ExtrAvidin-HRP (Sigma Aldrich) 1:10,000 and goat antimouse HRP-conjugated (BioRad) 1:1000. For AAL inhibition, the biotinylated lectin at its working dilution was incubated 30 min at RT with 0.2 M methyl-α-L-fucopyranoside prior to blot incubation. Blots were developed for detection by chemiluminescence (SuperSignal West Pico PLUS) using an ImageQuant LAS4000 imager (GE Healthcare), and quantification was performed using the ImageQuant TL software. The average of three biological repeats ±SD is shown.

Bandini G., Agop-Nersesian C., van der Wel H., Mandalasi M., Kim H.W., West C.M, & Samuelson J. (2020). The nucleocytosolic O-fucosyltransferase SPINDLY affects protein expression and virulence in Toxoplasma gondii. The Journal of Biological Chemistry, 296, 100039.

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Lectin-Based Glycan Analysis Protocol

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Biotinylated AAL, SNA, MAL-II, WGA, LCA, PSA, PNA, PHA-L, and GSL-1 lectins and 10x Carbo-free Blocking Buffer were purchased from Vector laboratories Inc. Biotinylated AOL lectin was purchased from TCI Europe. 0.2 mg/mL Streptavidin-phycoerythrin conjugate was purchased from Fischer Scientific (Invitrogen, eBioscience). Unnatural sugar derivatives were synthesized as described under synthetic procedures and stored at −20 °C at a concentration of 100 mM in DMSO.

Pijnenborg J.F., Rossing E., Merx J., Noga M.J., Titulaer W.H., Eerden N., Veizaj R., White P.B., Lefeber D.J, & Boltje T.J. (2021). Fluorinated rhamnosides inhibit cellular fucosylation. Nature Communications, 12, 7024.

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Enzyme Kinetics of SPY Fucosylation

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The in vitro enzyme activity of wild type and mutant SPY were analyzed using western blotting with biotinylated AAL described previously^{69 (link)} with slight modification. Briefly, for assessing the self-fucosylation of SPY, the reaction mixture consisted of 5 μg tag-removed enzyme, 60 μM GDP-fucose, 50 mM Tris pH 8.0, 300 mM NaCl, and 5 mM MgCl₂ in a total volume of 40 μl; for assessing the fucosylation of DELLA^1–205 by SPY, the reaction mixture consisted of 5 μg tag-removed enzyme, 20 μg DELLA^1–205, 60 μM GDP-fucose, 50 mM Tris pH 8.0, 300 mM NaCl, and 5 mM MgCl₂ in a total volume of 40 μl. The reactions were undertaken at room temperature for 1 h and then quenched at 95 °C for 90 s. Eight microliters of the reaction buffer was subjected to SDS-PAGE and blotted using biotinylated AAL (Vector Labs, cat# B-1395) with a dilution of 1:1000 and HRP-labeled Streptavidin (Sangon, cat# B110053) with a dilution of 1:5000 as the primary and secondary antibodies, respectively. Blots were developed for detection by ChemiScope5600 (Clinx).

Zhu L., Wei X., Cong J., Zou J., Wan L, & Xu S. (2022). Structural insights into mechanism and specificity of the plant protein O-fucosyltransferase SPINDLY. Nature Communications, 13, 7424.

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Glycan Profiling of Human Lung Tissues

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The human lung homogenate was prepared by blending the tissues in lysis buffer (25 mM TRIS, 150 mM NaCl, 5 mM EDTA, 1% CHAPS, pH 7.4), followed by sonication. Digestion of the homogenate by PNGase F, neuraminidase A and neuraminidase S was performed at 37 °C for 16 hours (New England Biolabs, Inc.). Proteins were stained with Coomassie Brilliant Blue (CBB) or transferred to a nitrocellulose membrane (Thermo Fisher Scientific). After blocking with 5% (w/v) bovine serum albumin (BSA) in TRIS-buffer saline with 0.05% TWEEN 20 (TBST) for 1 h at room temperature, Western and lectin blots were analyzed with anti-LeA (7LE, Santa Cruz Biotechnology), anti-SLeA (9L426, US Biological), anti-LeX (HI98, BioLegend, Inc.), biotinylated AAL, ConA, DBA, MAL-I, SNA, or UEA-I (Vector Laboratories) as a primary staining, and horseradish peroxidase (HRP)-labelled goat anti-mouse IgG antibody, goat anti-mouse IgM antibody, or streptavidin at 1:5000 dilution in TBST, using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).

Jia N., Byrd-Leotis L., Matsumoto Y., Gao C., Wein A.N., Lobby J.L., Kohlmeier J.E., Steinhauer D.A, & Cummings R.D. (2020). The Human Lung Glycome Reveals Novel Glycan Ligands for Influenza A Virus. Scientific Reports, 10, 5320.

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Western Blot and Lectin Blot Analysis

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Proteins separated by SDS-PAGE were transferred to PVDF membrane. The membrane was incubated with blocking buffer [5% non-fat milk in TBST (Tris buffered saline with 0.1% Tween-20, pH 7.5)] for 1 h at room temperature (RT). The blocked membrane was incubated with HRP-anti-biotin antibody (Jackson ImmunoResearch, West Grove, PA, United States) (1:100,000) or an HRP-anti-His (27E8) antibody (Cell Signaling Technology, Danvers, MA, United States) (1:1000) in blocking buffer overnight at 4°C, washed three times with TBST and visualized with ECL Western blotting detection reagents (Thermo Fisher Scientific, Waltham, MA, United States). Odyssey FC imaging system (LI-COR, United States) was used to detect the chemiluminescence. For Aleuria Aurantia Lectin (AAL) blot, the membrane was blocked in 5% bovine serum albumin (BSA) (Sigma, St. Louis, MO, United States) for 1 h at RT and incubated with biotinylated-AAL (Vector Laboratories, Burlingame, CA, United States) (1:500) in PBS for 1 h at RT. After being washed for three times with TBST, the membrane was incubated with HRP-anti-biotin antibody and visualized.

Tian X., Jiang H., Cai B., Feng H., Wang X, & Yu G. (2022). Comparative Proteomic Analysis of Fucosylated Glycoproteins Produced by Bacteroides thetaiotaomicron Under Different Polysaccharide Nutrition Conditions. Frontiers in Microbiology, 13, 826942.

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Quantification of Antibody Fucosylation

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Equal amounts of purified antibody were separated in reducing SDSpolyacrylamide gel electrophoresis, followed by transfer to polyvinylidene difluoride membranes. After blocking with tris-buffered saline with 0.1% Tween 20 + 3% bovine serum albumin (BSA), we incubated the membranes with either biotinylated AAL (200 ng/ml; Vector Laboratories) overnight at 4°C, followed by a 1-hour incubation with horseradish peroxidase (HRP)-conjugated streptavidin (20 ng/ml; Jackson ImmunoResearch), or as control overnight at 4°C with an HRP-conjugated goat anti-rat IgG (20 ng/ml; Jackson ImmunoResearch). Bound HRP was detected using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).
The surface fucosylation of the GK1.5 hybridoma was analyzed by staining in Hanks' balanced salt solution + 0.5% BSA with biotinylated AAL for 30 min at 4°C, followed by a 30-min incubation with allophycocyanin (APC)-labeled streptavidin (Thermo Fisher Scientific). Samples were then acquired on a Canto II flow cytometer, and mean fluorescence intensities were determined.

Wieland A., Kamphorst A.O., Valanparambil R.M., Han J.H., Xu X., Choudhury B.P, & Ahmed R. (2018). Enhancing FcγR-mediated antibody effector function during persistent viral infection. Science immunology, 3(27).

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