The protocol for native gel electrophoresis was an adaptation of previously described methods (39 (link), 72 (link)). The electrophoretic system was comprised of an upper 4% and a lower 8% acrylamide gel (37.5:1 acrylamide/bisacrylamide) containing buffer (30 mM β-alanine [Sigma] and 20 mM lactic acid [Sigma]) that was adjusted to pH 4.4 and 3.8, respectively. The buffers were identical to those used in the respective upper and lower gel compartments of the Mini-Protean III cell (Bio-Rad). Three types of htau40 monomers at 5 μM concentration were employed: reduced, oxidized, and oxidized incubated for 1 h with 20 mM DTT. The proteins were mixed with 2.5× sample buffer (0.01% methyl green [Sigma], 25% glycerol, 75 mM β-alanine, and 55 mM lactic acid) and then loaded onto the gel. The gel was run for 2 h at 250 V with polarity reversed and then stained with Coomassie blue for analysis.
β alanine
Manufactured by Merck Group
Sourced in United States, Spain, Germany, Czechia, Italy
β-alanine is a non-essential amino acid that serves as a precursor to the neurotransmitter carnosine. It is commonly used in laboratory settings for research and analysis purposes.
Automatically generated - may contain errors
Lab products found in correlation
Glycine, by Merck Group (10 mentions)
Sarcosine, by Merck Group (5 mentions)
Histamine, by Merck Group (5 mentions)
Taurine, by Merck Group (5 mentions)
Proline, by Merck Group (4 mentions)
Lactic acid, by Merck Group (4 mentions)
Citric acid, by Merck Group (4 mentions)
Acetonitrile, by Thermo Fisher Scientific (4 mentions)
Urea, by Merck Group (3 mentions)
Histidine, by Merck Group (3 mentions)
Chloroquine, by Merck Group (3 mentions)
Xylitol, by Merck Group (3 mentions)
Choline chloride, by Merck Group (3 mentions)
Acetonitrile, by Merck Group (3 mentions)
Methanol, by Merck Group (3 mentions)
Citrulline, by Merck Group (3 mentions)
Phenethylammonium iodide, by Xi'an Yuri Solar Co. (3 mentions)
Lead iodide pbi2, by Xi'an Yuri Solar Co. (3 mentions)
Dms c21, by Gelest (3 mentions)
Isoguvacine, by Merck Group (2 mentions)
58 protocols using β alanine
1
Native Gel Electrophoresis of Tau Protein
2
Taurine Depletion and Light Exposure
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Two-month-old albino Sprague-Dawley (SD) female rats (150-180 g body weight) were obtained from the University of Murcia breeding colony (n ¼ 40) and divided into a control group of rats drinking b-alanine-free water (intact group; n ¼ 20) and a group of rats treated with b-alanine (Sigma-Aldrich, Madrid, Spain) administered in the drinking water at a concentration of 3% to induce taurine depletion (Fig. 1; n ¼ 20). Animals were housed in an environmentally controlled room with a 12-hour:12-hour light-dark cycle (light from 8 AM to 8 PM) and had food and water ad libitum. The light intensity within the cages in our animal care facilities ranged from 5 to 30 lux. One month after the beginning of the treatment, half of the animals from both groups were exposed to light (see below). Light-exposed and nonexposed animals were processed 2 months after the beginning of the experiment, and 1month ALE (Fig. 1).
All experiments were carried out in accordance with the Spanish and the European Community Council Directives (86/ 609/EEC), and with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and were previously approved by the Ethics and Animal Studies Committee of the University of Murcia.
All experiments were carried out in accordance with the Spanish and the European Community Council Directives (86/ 609/EEC), and with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and were previously approved by the Ethics and Animal Studies Committee of the University of Murcia.
3
Measuring Calcium Flux in CHO Cells
4
C2C12 Myoblast Differentiation and Beta-Alanine Exposure
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C2C12 mouse myoblast from ATCC (Manassas, VA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4500mg/L glucose and supplemented with 10 % heat-inactivated fetal bovine serum (FBS) and 100U/mL penicillin/streptomycin, in a humidified 5% CO2 atmosphere at 37°C. Cells were seeded overnight and grown to confluence with growth media changed every two days. Differentiation was accomplished by replacing growth media with DMEM supplemented with 2% horse serum and 100U/mL penicillin/streptomycin for 6 days. Stock β-alanine and D-alanine (serving as an iso-osmolar, isonitrogenous control) from Sigma (St. Louis, MO) were dissolved in culture media to a final concentration of 800 µM. This concentration was used because to our knowledge, 800µM is the highest documented plasma concentration of β-alanine achieved following supplementation in healthy volunteers15 (link).
5
Expressing and Analyzing GABA-ρ Receptors
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The subcloned human GABA-ρ1 cDNA into pcDNA1.1 (Invitrogen, San Diego, CA, USA) was generously donated by Dr. George Uh1 (National Institute for Drug Abuse, Baltimore, MD, USA). The subcloned human GABA-ρ2 cDNA into PKS (Invitrogen) was generously provided by Dr. Garry Cutting (Center for Medical Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA).
GABA, muscimol, β-alanine, 5-amino valeric acid, glycine, isoguvacine, and imidazole-4-acetic acid were purchased from Sigma-Aldrich (Sigma-Aldrich Pty Ltd., Castle Hill, NSW 1765, Australia). TACA and CACA were prepared as previously reported [3 (link)].
GABA, muscimol, β-alanine, 5-amino valeric acid, glycine, isoguvacine, and imidazole-4-acetic acid were purchased from Sigma-Aldrich (Sigma-Aldrich Pty Ltd., Castle Hill, NSW 1765, Australia). TACA and CACA were prepared as previously reported [3 (link)].
6
Characterization of Organic Compounds
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All chemicals and reagents were of analytical grade, including glycolic acid (Acros Organics, Thermo Fisher Scientific, Branchburg, NJ, USA, #15451-0250), L-lactic acid (TCI, Zwijndrecht, Belgium, #L0165), 3-hydroxy propanoic acid (Sigma-Aldrich, St. Louis, MO, USA, #792659), (S)-3-hydroxy butyric acid (Sigma-Aldrich, St. Louis, MO, USA, #54925), glycine (BioRad, Hercules, CA, USA, #161-0717), L-alanine (Alfa Aesar, Haverhill, MA, USA, #A15804), β-alanine (Sigma-Aldrich, St. Louis, MO, USA,, #05159), and (S)-3-amino butyric acid (Sigma-Aldrich, St. Louis, MO, USA, #757454).
7
Recombinant Expression of Human GABA Receptor
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Human ρ1 cDNA subcloned into pcDNA1.1 (Invitrogen, San Diego, CA, USA) was kindly provided by Dr. George Uh1 (National Institute for Drug Abuse, Baltimore, MD, USA).
(GABA, 5-aminovaleric acid, β-alanine, glycine, isoguvacine, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) also known as Gaboxadol, were purchased from Sigma-Aldrich (Sigm-Aldrich Pty Ltd, Castle Hill, NSW 1765 Australia). 1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) [31 (link)], trans-aminocrotonic acid (TACA) and cis-aminocrotonic acid (CACA) [32 (link)], and 2-aminoethylmethane thiosulfonate (MTSEA) [33 (link)] were prepared as previously reported.
(GABA, 5-aminovaleric acid, β-alanine, glycine, isoguvacine, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) also known as Gaboxadol, were purchased from Sigma-Aldrich (Sigm-Aldrich Pty Ltd, Castle Hill, NSW 1765 Australia). 1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) [31 (link)], trans-aminocrotonic acid (TACA) and cis-aminocrotonic acid (CACA) [32 (link)], and 2-aminoethylmethane thiosulfonate (MTSEA) [33 (link)] were prepared as previously reported.
8
Amino Acid Synthesis Protocol
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Sodium acetate trihydrate (>99 wt%) was purchased from Scharlau (Sentmenat, Spain). Urea (>99.5 wt%), glycine (>99 wt%), β-alanine (>99 wt%), trans-4-hydroxy-L-proline (>99 wt%), L-lysine monohydrochloride (>99.5 wt%), L-arginine (>98 wt%), and L-cystine (>98 wt%) were supplied by Sigma-Aldrich (Madrid, Spain). L-Proline, with purity >99 wt%, was supplied by Panreac (Castellar del Vallès, Spain). Table S2 in Supplementary Materials shows sources and purities of the amino acids used in this work. All reactants were used as received, without further purification. Bidistilled water was used throughout the experimental work in preparing the aqueous solutions.
9
Comparative Study of Breast Cell Lines
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Non-malignant breast epithelial cells (MCF-10a) and malignant breast epithelial cells (MCF-7) were purchased from ATCC (Manassas, VA). MCF-10a cells were cultured in combination F-12 DMEM media supplemented with 50 nM hydrocortisone, 20 ng/ml EGF, 0.01 mg/ml human insulin, 5% fetal bovine serum (FBS), and 1% Pen-Strep. MCF-7 cells were cultured in DMEM supplemented with 10% FBS with 0.01 mg/ml human insulin, and 1% Pen-Strep. Cells were treated with β-alanine from Sigma (St. Louis, MO) at 100 mM dissolved in media and incubated in 5% CO2 at 37°C for 24 hours.
10
Untargeted Metabolomics Mass Spectrometry
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All solvents for untargeted mass spectrometry were LC-MS grade. Acetonitrile, methanol, and formic acid were purchased from Fisher Scientific (Fairlawn, New Jersey); Water was purchased from Honeywell (Muskegon Michigan); 1-methylhistidine, 3-methylhistidine, α-amino-n-butyric acid, alanine, anserine, arginine, asparagine, aspartic acid, β-aminoisobutryic acid, β-alanine, carnosine, citrulline, creatinine, cystathionine, cysteine, ethanolamine, γ-aminobutyric acid, glutamic acid, glutamine, glycine, histidine, homocystine, hydroxylysine, hydroxyproline, isoleucine, L-aminoadipic acid, L-cystine, leucine, lysine, proline, methionine, ornithine, phenylalanine, phosphoserine, phosphoethanolamine, sarcosine, serine, taurine, threonine, tryptophan, tyrosine, urea, and valine were purchased from Sigma Aldrich (St. Louis, Missouri); 10(S),17(S)-DiHDoHE (Protectin DX), 11β-PGF2α, 14(S)-HDHA, 15R-PGF2α, 17(S)-HDHA, 8-iso-15R-PGF2α, 8-iso-PGF2α, Lipoxin A4 (LXA4), LTB4, LTC4, LTD4, LTE4, PGE2, PGF2α, Resolvin D1 (RVD1), and Resolvin D2 (RVD2) were purchased from Cayman Chemical (Ann Arbor, Michigan).
All standards and deuterated internal standards used for LC-MS/MS analysis of arachidonic acid, docosahexaenoic acid derived lipid mediators were purchased from Cayman Chemical (Ann Arbor, Michigan, USA). All HPLC solvents and extraction solvents were HPLC grade or better.
All standards and deuterated internal standards used for LC-MS/MS analysis of arachidonic acid, docosahexaenoic acid derived lipid mediators were purchased from Cayman Chemical (Ann Arbor, Michigan, USA). All HPLC solvents and extraction solvents were HPLC grade or better.
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