Neisseria gonorrhoeae F62 was isolated from an uncomplicated infection (Kellogg, Peacock, Deacon, Brown, & Pirkel,
Erythrina cristagalli lectin eca
Manufactured by Vector Laboratories
Sourced in United States
Erythrina cristagalli lectin (ECA) is a carbohydrate-binding protein derived from the seeds of the coral tree (Erythrina cristagalli). ECA demonstrates specificity for galactose and N-acetylgalactosamine residues and is commonly used in biochemical and cell biology research applications.
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Lab products found in correlation
Sambucus nigra lectin sna, by Vector Laboratories (2 mentions)
Amicon ultra 0.5 ml centrifugal filter, by Merck Group (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Gemini Bio (1 mentions)
Isovitalex, by BD (1 mentions)
Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Maackia amurensis lectin 1 mal 1, by Vector Laboratories (1 mentions)
Octet red384, by Molecular Devices (1 mentions)
Phosphate buffered saline (pbs), by Lonza (1 mentions)
Neuraminidase from vibrio cholerae, by Merck Group (1 mentions)
β galactosidase from bovine testes, by Merck Group (1 mentions)
4 protocols using erythrina cristagalli lectin eca
1
Neisseria gonorrhoeae Infection Protocols
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Neisseria gonorrhoeae F62 was isolated from an uncomplicated infection (Kellogg, Peacock, Deacon, Brown, & Pirkel,1963 ). Neisseria gonorrhoeae 15253 was isolated from a disseminated infection (O'Brien, Goldenberg, & Rice, 1983 ). Both strains were piliated. The mutant strains of F62 (ΔlgtD, ΔlgtA, ΔlgtE, and ΔlgtF) were constructed using plasmids and methods described previously (Gulati et al., 2015 ). The LOS phenotype of the mutants was verified by LOS staining of protease K‐treated whole‐cell samples that were separated on a 12% Bis–Tris gel with MES running buffer. N. gonorrhoeae were grown overnight on chocolate agar plates with IsoVitaleX (BD Bioscience) or in GC broth supplemented with IsoVitaleX at 37°C and 5% CO2. When indicated, growth media was supplemented with 30 µM CMP‐Neu5Ac (Nacalai USA, Inc.). Incorporation of Neu5Ac has been confirmed by loss of Erythrina Cristagalli Lectin (ECA, Vector Laboratories), which binds to the lactosamine epitope. For all binding and infection studies, bacteria were cultivated to an optical density at 600 nm equivalent to 0.4–0.6. THP‐1 cells were grown in RPMI‐1640 (Gibco) with 10% fetal calf serum (Gemini Bio‐Products) at 37°C and 5% CO2.
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Neisseria gonorrhoeae F62 was isolated from an uncomplicated infection (Kellogg, Peacock, Deacon, Brown, & Pirkel,
2
Lectin Staining of NTHi with Sialic Acid
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NTHi grown in the presence or absence of sialic acid was used for lectin staining. Following Sia feeding, the bacterial culture (OD600 = 0.1) was spun at 10,000 relative centrifugal force (rcf) for 5 min and washed with HBSS containing CaCl2/MgCl2. The bacteria were incubated with biotinylated lectins (from Vector Laboratories)—Erythrina cristagalli lectin (ECA) and Maackia amurensis lectin I (MAL I)—at 37°C for 30 min. For 3F11 antibody (generous gift from Michael Apicella, University of Iowa) staining, the bacteria were incubated at room temperature for 30 min. The lectins were used at the following concentration in HBSS with 0.1% BSA-ECA at 1:500 (stock, 5 mg/ml), MAL I at 1:500 (stock, 2 mg/ml) and 3F11 at 1:50 dilution. The binding was detected with either streptavidin conjugated with Cy5 (Jackson ImmunoResearch Laboratories) (1:1,000) or anti-mouse antibody conjugated with fluorescein isothiocyanate (FITC) (Invitrogen) (1:1,000) and visualized using a BD FACSCalibur flow cytometer. The data were analyzed using FlowJo software.
3
Real-time virus binding analysis by BLI
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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)].
4
Glycan Modification of Antibodies
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For desialylation, 10 µg of AK23 mAb was incubated with 52.5 mU of neuraminidase from Vibrio cholerae (Sigma) for 120 h at 37 °C in a 50 mM sodium acetate buffer (pH 5). Enzyme and buffer were removed using Amicon Ultra 0.5 mL centrifugal filters with cut-off of 100 kDa (Merck Millipore, Burlington, MA, USA). Fifty micrograms of patient’s IgG was incubated with 37.5 mU for 120 h at 37 °C in a 50 mM sodium acetate buffer (pH 5). Enzymatic digestion efficiency was tested by Eastern blot using biotinylated conjugated Maackia amurensis lectin II (MAL II, Bioworld, Dublin, OH, USA) or Sambucus nigra lectin (SNA, EBL, Vector Laboratories, Inc, Burlingame, CA, USA).
For degalactosylation, 10 µg of AK23 mAb was incubated with 28 mU of β-galactosidase from bovine testes (Sigma) for 72 h at 37 °C in a 50 mM sodium acetate buffer (pH 5) (complete digestion). Enzyme and buffer were then removed using Amicon Ultra 0.5 mL centrifugal filters with cut-off of 100 kDa (Merck Milipore, Burlington, MA, USA). Enzymatic digestion efficiency was tested by Eastern blot using biotinylated conjugated Erythrina cristagalli lectin (ECA, Vector Laboratories, Inc, Burlingame, CA, USA).
For degalactosylation, 10 µg of AK23 mAb was incubated with 28 mU of β-galactosidase from bovine testes (Sigma) for 72 h at 37 °C in a 50 mM sodium acetate buffer (pH 5) (complete digestion). Enzyme and buffer were then removed using Amicon Ultra 0.5 mL centrifugal filters with cut-off of 100 kDa (Merck Milipore, Burlington, MA, USA). Enzymatic digestion efficiency was tested by Eastern blot using biotinylated conjugated Erythrina cristagalli lectin (ECA, Vector Laboratories, Inc, Burlingame, CA, USA).
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