β mercaptoethanol

Manufactured by MP Biomedicals

Sourced in United States

β-mercaptoethanol is a reducing agent commonly used in various laboratory applications. It functions by breaking down disulfide bonds in proteins, thereby disrupting their structure and facilitating analysis or purification.

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Lab products found in correlation

16 protocols using β mercaptoethanol

Cytokine-Activated NK Cell Generation

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Cytokine-activated NK cells were generated from human and canine peripheral blood and mouse splenocytes. Human NK cells, canine PBMCs, and mouse splenocytes were each cultured in complete RPMI supplemented with recombinant human IL-2 at 1000 IU/mL for 7 days. Half-volume media changes were performed every 2-3 days to refresh media and replace IL-2. At time of collection, cells were vigorously pipetted and mechanically lifted from the plate surface to ensure collection of adherent cells. Cells were then washed in PBS and utilized for cell sorting. All cells were cultured in incubators at 37oC at 5% CO2 in complete RPMI media (RPMI 1640 media (Invitrogen Life Technologies) supplemented with 10% Nu-Serum (Corning Life Sciences), 2mM L-glutamine (Glutamax^®, Gibco), 1% non-essential amino acids (Gibco), 5x10-5M b-mercaptoethanol (MP Biomedicals), 1mM Hepes buffer (Corning Life Sciences), 1mM sodium pyruvate (Gibco), and 1% penicillin/streptomycin (Corning Life Sciences).

Gingrich A.A., Reiter T.E., Judge S.J., York D., Yanagisawa M., Razmara A., Sturgill I., Basmaci U.N., Brady R.V., Stoffel K., Murphy W.J., Rebhun R.B., Brown C.T, & Canter R.J. (2021). Comparative Immunogenomics of Canine Natural Killer Cells as Immunotherapy Target. Frontiers in Immunology, 12, 670309.

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Culturing and Extracting Protein from EGPE Cells

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EGPE is a cell line obtained from bovine E. granulosus pe G1 maintained at our laboratory. Cells from passages 35 to 40 were used for all experiments. Briefly, EGPE cells were grown in medium 199 (Sigma), 1 mM sodium pyruvate (sodium salt, extra pure, Anhedra, Beijing, China) and 78 μg/mL β-mercaptoethanol (Merck, Darmstadt, Germany) at pH 7.9 (37°C; CO₂:air; 5/95%) and EGPE cell colonies were performed in 2% agarose (20,000 cells/well) (Echeverría et al., 2010 (link)). Cells were grown in a liquid medium for 20 days and protein extracts were obtained as previously (Maglioco et al., 2019 (link)). Briefly, cells were washed five times with DPBS and incubated in lysis buffer (8 mmol/L CHAPS,MP Biomedicals, 10 mmol/L Tris –HCl, pH 8, 2 mmol/L EDTA, 0.1% B‐mercaptoethanol,MP Biomedicals, and 1/100 protease inhibitor cocktail, Sigma‐Aldrich), at 4°C for 2 hours. Samples were then frozen-thawed three times and spun down at 10 000 g.
Cell colony supernatants were obtained after 5 days of incubation. Debris was removed by centrifuging the supernatants three times (3000 rpm). Then, samples were stored in aliquots at -20°C until use.

Maglioco A., Agüero F.A., Valacco M.P., Valdez A.J., Paulino M, & Fuchs A.G. (2022). Characterization of the B-Cell Epitopes of Echinococcus granulosus Histones H4 and H2A Recognized by Sera From Patients With Liver Cysts. Frontiers in Cellular and Infection Microbiology, 12, 901994.

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Murine Dendritic Cell Activation Assay

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Recombinant murine granulocyte-macrophage colony stimulating factor (rmGM-CSF) and recombinant murine interleukin 4 (rmIL-4) were purchased from Peprotech (Rocky Hill, NJ, USA). LPS was purchased from Sigma (St. Louis, MO, USA). β-mercaptoethanol was obtained from MP Biomedicals LLC (Solon, OH, USA). Pam3csk4, poly (I:C), and CpG ODN1826 were purchased from InvivoGen (San Diego, CA, USA). FITC-, APC-, and PE-conjugated anti-murine CD4, CD11c, CD40, CCR7, CD80, CD86, and MHC-II antibodies were purchased from BioLegend (San Diego, CA, USA) and eBiosciences (New York, NY, USA). Antibodies against CD11c, TLR4, MyD88, and IκB were purchased from Biosynthesis Biotechnology Co. Ltd. (Beijing, China). Antibodies against p65 and p38 were obtained from Zhongshan Golden Bridge Co. Ltd. (Beijing, China). Antibodies against phospho-p65, phospho-IRF3, phospho-ERK1/2, and IRF3 were from Cell Signaling Technology (Danvers, MA, USA). Antibodies against phospho-p38 and ERK1/2 were purchased from Bioworld Technology (Nanjing, China). Anti-Tmod1 antibody was prepared by AbMax Biotechnology Co., Ltd. (Beijing, China). Antibodies against GAPDH and β-actin were obtained from Santa Cruz Biotech. (Santa Cruz, CA, USA). Tmod1 adenovirus and control adenovirus were constructed by SinoGeneMax Co. Ltd. (Beijing, China).

Liu X., Xia X., Wang X., Zhou J., Sung L.A., Long J., Geng X., Zeng Z, & Yao W. (2021). Tropomodulin1 Expression Increases Upon Maturation in Dendritic Cells and Promotes Their Maturation and Immune Functions. Frontiers in Immunology, 11, 587441.

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Characterization of HEK293 CRE/CREB Luciferase Assay

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HEK293 cells stably expressing
the CRE/CREB
luciferase reporter gene (BPS Bioscience #60515), RPMI medium (Corning
#10-040), KRB (Amsbio #KRB-1000), l-glutamine (Gibco #25030-081),
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Gibco #15630-080),
sodium pyruvate (Gibco #11360-070), β-mercaptoethanol (MP #806444), d-glucose (#G-7528), fetal bovine serum (Fisher #03600511),
penicillin/streptomycin (Corning #30-002-Cl), hygromycin B (Alfa Aesar
#J60681), lipofectamine 2000 (Invitrogen #11668027), VIPR peptide
agonist (Sigma #V3628), VIPR peptide antagonist (Sigma #SCP0260),
GLP-1R peptide agonist (Sigma #9416), 6-well cell culture plates (Ultra
Cruz #sc-204443), 96-well cell culture plates (Sigma #CLS9102), luciferase
cell culture lysis reagent (Promega #E1531), luciferase assay reagent
(Promega #E1501), and Ultra-Sensitive Rat Insulin Kit (Crystal Chem
#90060) were purchased from vendors. Rat GLP-1R plasmid (#14944)^{51 (link)} and VIPR1 plasmid (#51865)^{52 (link)} were purchased from Addgene, Flag-tagged Human GLP-1R and
Flag-tagged pCMV-N-Flag negative control vector were purchased from
vendors (Sino Biological Inc. #HG13944-NF and #CV061), and pcDNA3.1
vector and INS-1 832/13 cells were kindly provided by Dr. Xianxin
Hua (University of Pennsylvania).

Redij T., Chaudhari R., Li Z., Hua X, & Li Z. (2019). Structural Modeling and in Silico Screening of Potential Small-Molecule Allosteric Agonists of a Glucagon-like Peptide 1 Receptor. ACS Omega, 4(1), 961-970.

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Molecular Interactions in Tyrosine Kinase Signaling

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Primers were purchased from IDT (Coralville, IA) and used without further purification. HEPES was purchased from American Bio (Canton MA). β-mercaptoethanol was purchased from MP Biomedicals (Santa Ana, CA). Poly(6:2:5:1 Ala, Glu, Lys, Tyr) hydrobromide peptide, ATP, MgCl₂, complete protease inhibitor mixture tablets, and nickel-NTA chromatography resin were purchased from Sigma Aldrich (St. Louis, MO). DTT, NaCI, imidazole, Triton X-10, a Pierce 6X-histidine-tag antibody, an IgG-Cy3 antibody, and an IgG-Cy5 antibody were purchased from Thermo Fisher Scientific (Carlsbad, CA). Phospho-Tie2 antibodies (pTyr992 and pSer1119) were purchased from Cell Signaling Technology (Danvers, MA). [γ-³²P]-ATP was purchased from PerkinElmer (Waltham, MA).

Kennedy M.A., Xu Z., Wu Y, & Sohl C.D. (2019). A Tie2 kinase mutation causing venous malformations increases phosphorylation rates and enhances cooperativity. Biochemical and biophysical research communications, 509(4), 898-902.

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Culturing Human Embryonic Stem Cells

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Five human embryonic stem cell (hESC) lines (H1, H9, CT2, CT3, and CT4) obtained from the UConn Stem Cell Core were grown on irradiated mouse embryonic fibroblasts (MEFs) using hESC media containing DMEM with F12 (DMEM/F12, 1:1 ratio, ThermoFisher Scientific) supplemented with 20% Knockout Serum Replacer (ThermoFisher Scientific), 1X nonessential amino acids, 1 mM l-glutamine (ThermoFisher Scientific), 0.1 mM β-mercaptoethanol (MP Biomedicals; Santa Ana, CA), and 4 ng/ml of basic fibroblast growth factor (bFGF, Millipore-Sigma; Burlington, MA). Cells were passaged weekly. DNA was harvested from cultures that were 60–80% confluent.

Goetjen A., Watson M., Lieberman R., Clinton K., Kranzler H.R, & Covault J. (2020). Induced pluripotent stem cell reprogramming-associated methylation at the GABRA2 promoter and chr4p12 GABAA subunit gene expression in the context of alcohol use disorder. American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics, 183(8), 464-474.

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iPSC Generation from Caucasian Fibroblasts

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Fibroblasts were reprogrammed into iPSCs by the Stem Cell core at UConn Health using retroviral vectors to express five factors (OCT4, SOX2, KLF4, c-MYC, and LIN28) or Sendai virus to express four factors (OCT4, SOX2, KLF4, and c-MYC). Seventeen fibroblast lines were reprogrammed using retroviral vectors and 12 lines using Sendai virus once that protocol became available at the UConn Stem Cell core. iPSCs were grown on irradiated MEFs using hESC media containing DMEM with F12 (DMEM/F12, 1:1 ratio, ThermoFisher Scientific) supplemented with 20% Knockout Serum Replacer (ThermoFisher Scientific), 1X nonessential amino acids, 1 mM l-glutamine (ThermoFisher Scientific), 0.1 mM β-mercaptoethanol (MP Biomedicals), and 4 ng/ml of bFGF (Millipore-Sigma). All clones used in this study were derived from Caucasian fibroblast donors. We examined 69 iPSC clones (19 T/T, 27 T/C, and 23 C/C, genotyped at rs279858) generated from 29 donors (mean age = 42.9 (SD = 10.6)); including eight clones from three female donors. iPSC cells were passaged weekly and DNA was harvested from cultures once they had reached 60–80% confluency.

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CRE/CREB Luciferase Reporter Assay

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HEK293 cell stably expressing CRE/CREB luciferase reporter gene (BPS Bioscience #60515), RPMI medium (Corning #10-040), Krebs-Ringer Solution, HEPES-buffered (Alfa Aesar #J67795-K2), L-Glutamine (Gibco #25030-081), HEPES (Gibco #15630-080), Sodium Pyruvate (Gibco #11360-070), β-mercaptoethanol (MP #806444), D-Glucose (#G-7528), Fetal Bovine Serum (Fisher #03600511), penicillin/streptomycin (Corning #30-002-Cl), Hygromycin B (Alfa Aesar #J60681), Lipofectamine 2000 (Invitrogen #11668027), vasoactive intestinal peptide receptor (VIPR) peptide agonist (Sigma #V3628), VIPR peptide antagonist (Sigma #SCP0260), GLP-1R peptide agonist (Sigma #9416), 6-well cell culture plates (Ultra Cruz #sc-204443), 96-well cell culture plates (Sigma #CLS9102), Luciferase cell culture lysis reagent (Promega #E1531), Luciferase assay reagent (Promega #E1501), and Ultra-Sensitive Rat Insulin Kit (Crystal Chem #90060) were purchased from vendors. VIPR1 receptor plasmid (#51865) was purchased from Addgene, Flag-tagged Human GLP-1R and Flag-tagged pCMV-N-Flag negative control vector were purchased from vendors (Sino Biological Inc. #HG13944-NF and #CV061) and INS-1 832/13 cells were provided by Dr. Xianxin Hua at Perelman School of Medicine, Pfu DNA polymerase kit (Thermofisher #EP0501) and primers (University of Pennsylvania) were purchased from vendors.

Redij T., McKee J.A., Do P., Campbell J.A., Ma J., Li Z., Miller N., Srikanlaya C., Zhang D., Hua X, & Li Z. (2022). 2-Aminothiophene derivatives as a new class of positive allosteric modulators of glucagon-like peptide 1 receptor. Chemical biology & drug design, 99(6), 857-867.

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RNA Extraction and Quantification Protocol

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Cells were washed with DPBS and then lysed with either RLT lysis buffer (Qiagen, Hilden, Germany) supplemented with β-mercaptoethanol (MP Biomedicals, Santa Ana, CA, USA) or QIAzol lysis reagent (Qiagen) before collection by scraping. RNA was extracted using either RNeasy micro kit for RLT-lysed samples or miRNeasy micro kit for QIAzol-lysed samples (both from Qiagen), according to the instructions of the manufacturer with DNase treatment included. RNA quality was assessed using either Agilent 6000 Nano Kit on a 2100 BioAnalyzer instrument or DNF-471 RNA kit on a Fragment Analyzer (all from Agilent Technologies, Santa Clara, CA, USA), according to the instructions of the manufacturer. Concentrations were quantified with Qubit 4 Fluorometer using the Qubit RNA HS Assay Kit (Invitrogen, Thermo Fisher Scientific). RNA was stored at −80°C.

Dolatabadi S., Jonasson E., Andersson L., Luna Santamaría M., Lindén M., Österlund T., Åman P, & Ståhlberg A. (2022). FUS-DDIT3 Fusion Oncoprotein Expression Affects JAK-STAT Signaling in Myxoid Liposarcoma. Frontiers in Oncology, 12, 816894.

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Expansion of Mouse Splenocytes with IL-2

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Mouse splenocytes were cultured in complete RPMI supplemented with recombinant human IL-2 at 1000 IU/mL for 7 days. Half-volume media changes were performed every 2-3 days to refresh media and replace IL-2. All cells were cultured in incubators at 37°C at 5% CO2 in complete RPMI media (RPMI 1640 media (Invitrogen Life Technologies) supplemented with 10% Nu-Serum (Corning Life Sciences), 2mM L-glutamine (Glutamax^®, Gibco), 1% non-essential amino acids (Gibco), 5x10-5M β-mercaptoethanol (MP Biomedicals), 1mM Hepes buffer (Corning Life Sciences), 1mM sodium pyruvate (Gibco), and 1% penicillin/streptomycin (Corning Life Sciences).

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