Lectin ELISA was performed on purified IgG, as has been published with plasma samples [6 (link)]. Briefly, purified IgG was diluted to 1 mg/mL in carbonate coating buffer (100 mM NaHCO3, 30 mM NaCO3, pH 9.5), pipetted into a 96-well high-binding ELISA plate (Microlon High Binding; Greiner BioOne), and incubated overnight at 4 °C. The plate was blocked with carbohydrate free blocking solution (Vector Labs) for 1 hour at room temperature. Biotinylated lectins (Vector Labs) were diluted to 1 μg/mL in carbohydrate free blocking solution and incubated on the plate for 1 hour at room temperature. Signal was detected using europium-conjugated streptavidin (Perkin Elmer) and time-resolved fluorescence as measured in a Victor V3 1420 multilabel plate reader.
Carbohydrate free blocking solution
Manufactured by Vector Laboratories
Sourced in United States
Carbohydrate-free blocking solution is a laboratory reagent designed to prevent non-specific binding in various immunoassays and blotting techniques. It is a protein-based solution that does not contain any carbohydrates.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Complete ultra protease inhibitor tablet, by Roche (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Fluorescein conjugated lectins, by Vector Laboratories (1 mentions)
Biotinylated lectin, by Vector Laboratories (1 mentions)
Europium conjugated streptavidin, by PerkinElmer (1 mentions)
Microlon high binding, by Greiner (1 mentions)
Forskolin, by Merck Group (1 mentions)
Ro 20 1724, by Merck Group (1 mentions)
Skim milk powder, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
2 fluoro peracetyl fucose 2f fuc, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Selleck Chemicals (1 mentions)
6 protocols using carbohydrate free blocking solution
1
Lectin ELISA for Purified IgG
2
Lectin Profiling of Cultured Cells
3
Lectin Histochemistry Protocol for Glomerular Analysis
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For lectin histochemistry, fluorescein isothiocyanate (FITC)‐labeled lectins and wheat germ agglutinin (WGA) were purchased from EY Laboratories (San Mateo, CA, USA). Paraffin‐embedded sections were deparaffinized, rehydrated, and blocked in carbohydrate‐free blocking solution (Vector Laboratories, Burlingame, CA, USA). The slides were incubated at 4 °C overnight with lectin aliquoted in carbohydrate‐free blocking solution (FITC–WGA, 15 µg·mL−1). Washes were performed with PBS [21 ]. We randomly selected five glomeruli per mouse and measured the luminance of them in each experimental group using KS 400.
4
Molecular Reagents for Cell Signaling
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Dimethyl sulfoxide (DMSO) was purchased from Sigma. (1R,2R)-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4) was a gift from Prof. Ronald Schnaar (Johns Hopkins); stock concentrations were made at 5 mM in water. Bovine serum albumin (BSA) was purchased from Sigma. Skim milk powder was purchased from Fisher Scientific (catalog no. NC9121673). Carbohydrate-free blocking solution was purchased from Vector Laboratories (Burlingame, CA) (catalog no. SP-5040). COmplete ULTRA protease inhibitor tablets were purchased from Roche. Brefeldin A (≥ 99% pure) was purchased from Sigma (catalog no. B7651); stock concentrations were made at 5 mg/ml in DMSO. Forskolin (≥ 98% pure) was purchased from Sigma (catalog no. F6886); stock concentrations were made at 10 mM in DMSO. 1-Methyl-3-Isobutylxanthine (IBMX, ≥ 98% pure) was purchased from Cayman Chemicals (catalog no. 13347); stock concentrations were made at 50 mM in DMSO. Ro 20-1724 [4-(3-butoxy-4-methoxy-benzyl) imidazolidone] was purchased from Sigma Aldrich (catalog no. B8279); stock concentrations were made at 100 mM in DMSO.
5
Reagents for Cellular Assays
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Dimethyl sulfoxide (DMSO) was purchased from Sigma. 2-Fluoro-peracetyl- fucose [2F-Fuc; 98.8% pure] was purchased from EMD Millipore (Darmstadt); stock concentrations were made at 200 mM in DMSO. Niclosamide [niclo] was purchased from Selleckchem; stock concentrations were made at 5 mM in DMSO. Bovine serum albumin [BSA] was purchased from and Sigma. Carbohydrate-free blocking solution was purchased from Vector Laboratories. Complete ultra protease inhibitor tablets were purchased from Roche.
6
Quantifying Cell Death and Lectin Binding
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A549 cells were harvested after stimulation and stained with SYTOX live/dead stain as well as a panel of FITC-conjugated lectins diluted in Carbohydrate-free blocking solution (Vector Laboratories, Newark, CA). Cells were analyzed using the Attune NxT in the Cytometry and Imaging Microscopy Shared Resource of the Case Comprehensive Cancer Center. Flow data was analyzed using FlowJo Software (BD Biosciences, Franklin Lakes, New Jersey). Cell death was calculated by the ratio of SYTOX positive and negative cells among single cells. Lectin mean fluorescence intensity was determined among the SYTOX negative (i.e. alive) population, thereby excluding dead cells from the analysis.
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