1-Palmitoyl-2-oleoyl-sn-phosphatydilhcoline (POPC), (Molecular weight: 760.08) and monosialoganglioside-GM1 (Molecular weight: 1545) from bovine brain and Chol (Molecular weight: 386.66) were purchased from SIGMA (SIGMA Chemical, St Louis, MO). Lyophilized sCT (Molecular weight: 3432) was purchased from European PHARMACOPOEIA (EDQM, Strasbourg, France) and stored at −18 °C before use. Chloroform, methanol, and other solvents were also purchased from SIGMA. Stock solution of POPC was prepared in Chloroform (1 mg/ml). sCT was dissolved in 4:1 (v/v) Chloroform/methanol containing 0.5% amyl alcohol. All the chemicals were used without further purification. Phosphate Buffered Saline (PBS, pH 7.4, I 0.16) was used. In all experiments and for cleaning procedures only ultra-pure MILLI-Q water was used. The composition of lipid-rafts was simulated using different mixtures of GM1, Chol and POPC. Chol and POPC were dissolved in Chloroform and GM1 in 2:1:0.15 (v/v) Chloroform/methanol/water. All used concentrations were 1 mg/ml. These solutions were mixed in appropriate ratios to form the different model membranes: i.e. GM1/Chol/POPC molar ratios 0.50/0.25/0.25: Chol/POPC 0.50/0.50 molar ratio. sCT was added to the lipid mixture before the Langmuir film preparations.
Monosialoganglioside gm1
Manufactured by Merck Group
Sourced in United States
Monosialoganglioside-GM1 is a laboratory product manufactured by Merck Group. It is a glycosphingolipid compound that can be used in various research applications. The core function of Monosialoganglioside-GM1 is to serve as a reference standard or analytical tool for researchers studying gangliosides and related biomolecules.
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Lab products found in correlation
Cholera toxin, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Chloroform, by Merck Group (1 mentions)
Elisa plate, by Corning (1 mentions)
Bovine serum albumin (bsa), by Nacalai Tesque (1 mentions)
Ap substrate, by Merck Group (1 mentions)
Immuno mini nj 2300, by Thermo Fisher Scientific (1 mentions)
96 well microtiter plates, by Sumitomo Bakelite (1 mentions)
Pencillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Anti cholera toxin antibody, by Merck Group (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg peroxidase, by Merck Group (1 mentions)
Ketamine hydrochloride, by Syntec (1 mentions)
Xylazine hydrochloride, by Syntec (1 mentions)
Carbachol, by Merck Group (1 mentions)
Prostaglandin e2, by Merck Group (1 mentions)
Ab34992, by Abcam (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
13 protocols using monosialoganglioside gm1
1
Lipid-Raft Simulation with Peptide
2
Quantifying LT Toxin Secretion in Bacteria
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LT toxin secretion by different strains was measured using previously established GM1-ELISA method [81 (link)]. Briefly, ELISA plates (Costar, Corning, NY) were coated with 100 μl/well of 1 μg/ml monosialoganglioside GM1 (Sigma, G-7641), and incubated overnight at RT. After blocking with 2% BSA in PBS-Tween, 100 μl of clarified culture supernatant from overnight Luria broth cultures of bacteria was added to wells in triplicate and incubated for 1 h at 37°C. The wells were then washed and incubated with 100 μl of a 1:5000 dilution of rabbit anti-LTB polyclonal antisera for 1 h. Plates were again washed and then incubated with 100 μl of a 1:5000 dilution of anti-rabbit secondary conjugated with HRP for another hour. Finally plates were developed with 100 μL per well of TMB-H2O2 (KPL) substrates and read immediately at 630nm for kinetic measurement (Eon, BioTek instruments, VT, USA).
3
GM1-ELISA for Bacterial Binding
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A GM1-ELISA was performed to determine specific GM1-ganglioside-binding activities. Briefly, 96-well microtiter plates (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) were coated with 5 μg/ml monosialoganglioside GM1 (Sigma-Aldrich) diluted in bicarbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6), and incubated overnight at 4°C. After incubation, the plates were washed three times with PBS supplemented with 0.05% Tween-20 (PBS-T), and then blocked with PBS containing 1% bovine serum albumin (Nacalai Tesque, Kyoto, Japan) at 37°C for 2 h. After the plates were washed, the concentrated supernatant of L. casei (100 μl/well), rCTB–YVAD (50 ng/well), or rCTB (50 ng/well) was applied to the wells and incubated at 37°C for 2 h. The plates were incubated at 37°C for 2 h with antibody directed against CT and AP-labeled anti-rabbit IgG antibody used as the primary and secondary antibodies, respectively. The plates were incubated with AP substrate (Sigma-Aldrich) at 37°C for 20 min, and the OD405 was then measured with a microplate reader (ImmunoMini Nj-2300; Nunc, Rochester, NY).
4
Neuroprotective Effects of Pharmacological Interventions in Hypobaric Hypoxia
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A total of 30 healthy male Sprague-Dawley rats (age, 6–7 weeks; weight, 200–310 g) were purchased from the Experimental Animal Center of Wenzhou Medical College (Wenzhou, China). Rats were housed under a 12 h light/dark cycle at a controlled temperature of 22–25°C and a humidity of 55±5%, with ad libitum access to water and food throughout the study. After acclimatization to the laboratory environment for 1–2 days, the rats were randomly divided into five groups, as follows (6 rats/group): i) Control (CK) group, in which the rats were raised under normal laboratory conditions; ii) HH group, in which the rats were exposed to HH conditions without drug treatment; iii) CU group (Sigma-Aldrich, St. Louis, MO, USA), in which the rats were exposed to HH conditions and were subsequently treated with CU; iv) dimethyl sulfoxide (DMSO; Sigma-Aldrich) group, in which the rats were exposed to HH conditions and were subsequently injected with DMSO; and v) monosialoganglioside (GM1; Sigma-Aldrich) group, in which the rats were exposed to HH conditions and were treated with GM1. The present study was approved by the Medical Ethics Committee of Wenzhou Medical College, and all procedures were in compliance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications no. 80–23).
5
Silkworm-Derived GM1 Affinity Assay
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A GM1-ELISA was performed as described previously [13 (link)] to detect the affinity of silkworm-derived fusion proteins for GM1 ganglioside. The microtiter plates were coated with monosialoganglioside-GM1 (Sigma, USA) by incubating the plates with 50 μl/well GM1 in methanol at 4°C overnight. The wells were then blocked with BSA solution, and the hemolymph dilutions were added. The same dilutions of normal hemolymph and serial dilutions of bacterial CTB were used as a negative control and to generate the standard curve, respectively. The remainder of the procedure was identical to the semiquantitative ELISA assay described above.
6
Cholera Toxin Binding Assay
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Cholera toxin, anti-Cholera toxin antibody, anti-rabbit IgG-peroxidase, monosialoganglioside GM1, o-Phenylenediamine were procured from Sigma Aldrich (St. Louis, MI, USA). CHO (Chinese Hamster Ovary) cell line was obtained from NCCS, Pune. Dulbecco’s Modified Eagle Medium (DMEM), Fetal Bovine Serum, pencillin-streptomycin, and amphotericin B were purchased from Thermo Fischer Scientific (Waltham, MA, USA).
7
Pharmacological Characterization of Gastrointestinal Agents
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The following materials were obtained from commercial sources: castor oil, prostaglandin E2, charcoal, atropine, carbachol (CCh), Splittgerber’s reagent, cholera toxin, monosialoganglioside-GM1, and 3,3’,5’,50-Tetramethylbenzidine, from Sigma-Aldrich, Inc. (St Louis, MO, USA); bethanechol chloride, loperamide hydrochloride, and naloxone hydrochloride from Janssen-Cilag Pharmaceutics LTDA, Brazil and CRISTÁLIA Pharmaceutical Chemicals products LTDA, Brazil; Xylazine hydrochloride and ketamine hydrochloride, were obtained from Syntec (Cotia, SP, Brazil). All other chemicals used were of analytical grade and obtained from standard commercial suppliers. All drugs were dissolved in saline or phosphate-buffered saline (PBS).
8
Cholera Toxin Antibody Procurement
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CT and monosialoganglioside GM1 were purchased from Sigma-Aldrich® (St. Louis, MO, USA). Rabbit polyclonal antibody against CT (ab123129) and CTB (ab34992) were obtained from Abcam (Cambridge, UK). Rabbit polyclonal antibody against CTA (AB-43) was purchased from Advanced Targeting Systems (San Diego, CA, USA).
9
Fluorescent Labeling of SV40 Viral Proteins
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Monosialoganglioside GM1 (containing α-5-N-acetyl-muraminic acid) from bovine brain and unlabeled cholera toxin B were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Alexa Fluor 488-labeled CTXB was purchased from Invitrogen (Carlsbad, CA). Rabbit anti-SV40 VP1 antibody was purchased from Abcam (Cambridge, MA). Monoclonal anti-SV40 VP1 polyclonal antibody (PAb) 597 and monoclonal anti-SV40 large T antigen PAb108 were harvested from cultured hybridoma cells. Neutralizing anti-SV40 horse antiserum was a kind gift of James Pipas, University of Pittsburgh. Lipofectamine RNAi Max reagent, SimplyBlue SafeStain, and SuperSignal West Pico chemiluminescent substrate were purchased from Thermo, Fisher (Grand Island, NY). A DNeasy kit was purchased from Qiagen (Valencia, CA). IScript cDNA synthesis and iQ SYBR green supermix kits were purchased from Bio-Rad (Hercules, CA). TaqMan gene expression master mix was purchased from Applied Biosystems (Foster City, CA).
10
Quantifying GM1 Levels and IgG1 Response
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The GM1 levels in the cortex, cerebellum, brain stem and hippocampus homogenates from WT, β-Gal−/− and treated β-Gal−/− mice were measured by enzyme-linked immunosorbent assay (ELISA). Briefly, immulon® 4HBX 96-well plates (Thermo Scientific, Waltham, MA, USA) were coated with the Cholera toxin B subunit for GM1 capture. After binding of GM1 content on lysate samples, GM1 was detected using anti-GM1 antibody (Abcam, Cambridge, UK, Cat# ab23943). The GM1 levels from samples were determined based on a standard curve of commercial monosialoganglioside-GM1 (Sigma, St. Louis, MO, USA, Cat# G7641) and the final concentration was normalized to nanogram of GM1 per microgram of total soluble proteins (ng of GM1/µg total soluble protein).
IgG1 serum concentration against the RTB-fusion product and RTB domain were measured by ELISA using the SBA Clonotyping System-C57BL/6-HRP (Southern Biotech, Birmingham, AL, USA, Cat#: 5300-05B) following the manufactured instructions. Briefly, plates were coated overnight with 2.5 μg/mL of mβ-Gal:RTB or RTB domain produced and purified in an analogous system. Serum samples were incubated at a 1:400 dilution for 1 h. Measured IgG1 levels were quantified using an IgG1 standard curve (Southern Biotech, Birmingham, AL, USA, Cat#: 5300-01).
IgG1 serum concentration against the RTB-fusion product and RTB domain were measured by ELISA using the SBA Clonotyping System-C57BL/6-HRP (Southern Biotech, Birmingham, AL, USA, Cat#: 5300-05B) following the manufactured instructions. Briefly, plates were coated overnight with 2.5 μg/mL of mβ-Gal:RTB or RTB domain produced and purified in an analogous system. Serum samples were incubated at a 1:400 dilution for 1 h. Measured IgG1 levels were quantified using an IgG1 standard curve (Southern Biotech, Birmingham, AL, USA, Cat#: 5300-01).
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