6 aza 2 thiothymine

Manufactured by Merck Group

Sourced in Germany, United Kingdom

6-aza-2-thiothymine is a chemical compound used in laboratory research. It is a derivative of thymine, a nucleic acid base found in DNA. The core function of this product is to serve as a chemical building block for researchers and scientists working in various fields, such as biochemistry, molecular biology, and drug discovery.

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6-AZA (2 citations)

5 protocols using 6 aza 2 thiothymine

Purification and Characterization of Recombinant Proteins

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Recombinant HUαβ heterodimer from Escherichia coli was purified in-house as previously described [9 (link)]. Alkaline protease-treated streptavidin from Streptomyces avidinii was purchased from Promega (Madison, WI, USA). Myoglobin, ubiquitin, cytochrome c, and biotin were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France). The matrices 4-hydroxy-α-cinnamic acid (HCCA), dihydroxybenzoic acid (DHB), and sinapinic acid (SA) were from Bruker Daltonics (Bremen, Germany). All other matrices, i.e., 9-aminoacridine (9AA), aniline (ANI), 2-nitrophenol (2NPHL), 6-aza-2-thiothymine (ATT), 3-aminoquinoline (3AQ), 4-aminoquinaldine (4AQA), and picolinic acid (PLA), were purchased from Sigma-Aldrich. Ammonium hydroxide was procured from Fluka (Steinheim, Germany), while diethanolamine (DEOA) and glycerol (Gly) were from Sigma. Ammonium acetate was from Merck (Darmstadt, Germany) and acetic acid from Thermo-Fisher Scientific (Illkirch, France). The organic solvents acetonitrile and methanol were purchased from Carlo Erba (Milan, Italy). All solvents and buffers were prepared using 18 MΩ water purified with Milli-Q reagent grade system from Millipore (Bedford, MA, USA), herein referred to as “ultrapure water.”

Beaufour M., Ginguené D., Le Meur R., Castaing B, & Cadene M. (2018). Liquid Native MALDI Mass Spectrometry for the Detection of Protein-Protein Complexes. Journal of the American Society for Mass Spectrometry, 29(10), 1981-1994.

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Monoisotopic MALDI-TOF MS Protocol for Glycan Analysis

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Monoisotopic MALDI-TOF MS was performed using an Autoflex Speed (Bruker Daltonics, Bremen) instrument in either positive or negative reflectron modes with 6-aza-2-thiothymine (ATT; Sigma-Aldrich) as matrix; samples (0.8 µl) were vacuum dried on ground or polished steel plates before addition of matrix (0.8 µl of 3 mg/ml ATT in 50% ethanol) and crystallisation again under vacuum (22 (link)). The matrix region was suppressed and so MS spectra were normally recorded in the range m/z 700-3500. MS/MS was in general performed by laser-induced dissociation of the [M+H]⁺ or [M-H]^- pseudomolecular ions; typically 1000 shots were summed for MS and 3000 for MS/MS. Spectra were processed with the manufacturer’s software (Bruker Flexanalysis 3.3.80) using the SNAP algorithm with a signal/noise threshold of 6 for MS (unsmoothed) and 3 for MS/MS (four-times smoothed). Glycan spectra were manually interpreted on the basis of the masses of the predicted component monosaccharides, differences of mass in glycan series, fragmentation pattern, comparison with co-eluting structures from other insects and nematodes and chemical or exoglycosidase digestions. The symbolic annotations of spectra and chromatograms are according to the standard nomenclature (25 (link)).

Stanton R., Hykollari A., Eckmair B., Malzl D., Dragosits M., Palmberger D., Wang P., Wilson I.B, & Paschinger K. (2017). The underestimated N-glycomes of lepidopteran species. Biochimica et biophysica acta, 1861(4), 699-714.

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Lipid Profiling by MALDI-TOF Mass Spectrometry

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The LC-MS-grade solvents used were acetonitrile, isopropanol (Sigma-Aldrich, St. Louis, MO, USA) was used as an additive for the mobile phase and MALDI matrix solution preparation. The ultrapure water was purified by a Direct-Q water system (Millipore, Bedford, MA, USA). The MALDI matrices were 2,5-dihydroxybenzoic acid (DHB), 2-mercaptobenzothiazole (MBT), 6-aza-2-thiothymine (ATT), 4-nitroaniline (4-NA), N,N,N′,N′-tetramethyl-1,8-naphthalenediamine (DMAN), 9-aminoacridine (9-AA) and trans-ferulic acid (Sigma-Aldrich). The lipid standards used for TOF mass calibration were sphingomyelin, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 2-oleoyl-1-palmitoyl-glycero-3-phosphocholine, and L-α-phosphatidylcholine (Sigma-Aldrich).

Galeano Garcia P., Neves dos Santos F., Zanotta S., Eberlin M.N, & Carazzone C. (2018). Metabolomics of Solanum lycopersicum Infected with Phytophthora infestans Leads to Early Detection of Late Blight in Asymptomatic Plants. Molecules, 23(12), 3330.

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HPLC-based Characterization of ATT and DHB

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Chemicals used were of a high purity grade and included the following: HPLC grade chloroform (Sigma-Aldrich, Dorset, UK), HPLC water (Sigma-Aldrich), HPLC grade methanol (Fisher Scientific Ltd., Loughborough, UK) and 99.99 % pure formic acid (VWR International, East Grinstead, UK). Two different matrices were used in this study: 6-aza-2-thiothymine (ATT) and 2,5-dihydroxybenzoic acid (DHB; both from Sigma-Aldrich).

AlMasoud N., Xu Y., Trivedi D.K., Salivo S., Abban T., Rattray N.J., Szula E., AlRabiah H., Sayqal A, & Goodacre R. (2016). Classification of Bacillus and Brevibacillus species using rapid analysis of lipids by mass spectrometry. Analytical and Bioanalytical Chemistry, 408(27), 7865-7878.

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Lipid Extraction and Analysis Protocol

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HPLC grade water and acetonitrile (ACN) were purchased from (VWR International-UK). Acetone was purchased from (Fisher Scientific, Loughborough, UK). HPLC grade methanol (MeOH, ≥99.9%), HPLC grade chloroform (CHCl₃, ≥99.9%), 5-chloro-2-mercaptobenzothiazole (5C2M), 2-mercaptobenzothiazole, 6-aza-2-thiothymine, 2-(4-hydroxyphenylazo) benzoic acid, 2,5-dihydroxy benzoic acid (DHB), super-DHB, sinapic acid, 2,4,6-trihydroxy acetophenone monohydrate, 2,5-dihydroxy acetophenone, α-cyano-4-hydroxycinnamic acid, picolinic acid, and 9-aminoacridine hemihydrate were purchased from (Sigma Aldrich, Gillingham, UK). Porcine Gb3 standard and N-heptadecanoyl ceramide trihexoside (C17:0) internal standard were purchased from Matreya (Pleasant Gap, PA, USA).

Alharbi F.J., Geberhiwot T., Hughes D.A, & Ward D.G. (2016). A Novel Rapid MALDI-TOF-MS-Based Method for Measuring Urinary Globotriaosylceramide in Fabry Patients. Journal of the American Society for Mass Spectrometry, 27, 719-725.

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