Bacterial alkaline phosphatase

Manufactured by Merck Group

Sourced in Germany

Bacterial alkaline phosphatase is an enzyme derived from bacterial sources. It is used as a laboratory reagent to catalyze the hydrolysis of phosphate groups from various substrates.

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9 protocols using bacterial alkaline phosphatase

Purification and Characterization of Biomolecules

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All chemicals and reagents used were obtained at the highest purity available and were used without further purification unless stated. Benzonase, bacterial alkaline phosphatase, butylated hydroxytoluene, acetonitrile, and buffer salts were purchased from Sigma-Aldrich. Coformycin was obtained from the National Cancer Institute. Phosphodiesterase I was purchased from Worthington. Tetrahydrouridine was purchased from Calbiochem. Water purified through a Milli-Q system (Millipore) was used throughout our studies. Sartorius Vivaspin 500-brand centrifugal filter units were used for dialysis and sample concentration.

Lin C.J., Smibert P., Zhao X., Hu J.F., Ramroop J., Kellner S.M., Benton M.A., Govind S., Dedon P.C., Sternglanz R, & Lai E.C. (2015). An extensive allelic series of Drosophila kae1 mutants reveals diverse and tissue-specific requirements for t6A biogenesis. RNA, 21(12), 2103-2118.

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Purification of Enzymes for Molecular Biology

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Bacterial alkaline phosphatase, nuclease P₁ from Penicillium citrinum, snake venom phophodiesterase I and DNase were purchased as lyophilized powders from Sigma and stored in 50% glycerol with the appropriate buffer at the recommended temperature. PfuUltra DNA polymerase was obtained from Agilent (Santa Clara, CA, USA). Restriction enzymes were purchased from Fermentas (Glen Burnie, MD, USA) and New England Biolabs (Ipswich, MA, USA). Lysozyme was purchased from RPI Corporation (Mount Prospect, IL, USA). The plasmid encoding the Δ(172–173) variant of T7 RNA polymerase [40 (link)] was provided by John Perona. Recombinant Methanocaldococcus jannashii TGT was over-produced and purified as described previously [41 (link)].

Bon Ramos A., Bao L., Turner B., de Crécy-Lagard V, & Iwata-Reuyl D. (2017). QueF-Like, a Non-Homologous Archaeosine Synthase from the Crenarchaeota. Biomolecules, 7(2), 36.

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Characterization of Glycosinolate Hydrolysis

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Micrococcus nuclease, proteinase K, RNase A and bacterial alkaline phosphatase were purchased from Sigma-Aldrich (Taufkirchen, Germany). Phosphodiesterase II from calf spleen was obtained from Merck Biosciences Ltd. (Darmstadt, Germany), nuclease P1 from MP Biochemicals LLC (Eschwege, Germany), T4 polynucleotide kinase from MBI Fermentas (St. Leon-Rot, Germany), and [γ-³²P]ATP from Hartmann Analytic (Braunschweig, Germany). GLs and myrosinase (from seeds of Sinapis alba L.) were generous gifts of Dr. Renato Iori, Bologna. They were prepared as described elsewhere (Baasanjav-Gerber et al. 2011b (link)). One myrosinase unit was defined as the amount of enzyme able to hydrolyse 1 μmol sinigrin per min at pH 6.5 and 37 °C.

Glatt H., Engst W., Florian S., Schreiner M, & Baasanjav-Gerber C. (2022). Feeding Brassica vegetables to rats leads to the formation of characteristic DNA adducts (from 1-methoxy-3-indolylmethyl glucosinolate) in many tissues. Archives of Toxicology, 96(3), 933-944.

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18S rRNA Nucleoside Profiling Protocol

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Seventy microgram of 18S rRNA purified on a velocity gradient, whose integrity had been checked by migration on a denaturing agarose gel, was first heated for 2 min on a heating block in an Eppendorf cup at 100°C. This was followed by rapid cooling on ice. Five microliter of 10-mM ZnS0₄ was next added to the cup, followed by 10-μl P1 nuclease (Sigma). Nuclease digestion was carried out at 37°C for 16 h (overnight). After 16 h, 10 μl of 0.5-M Tris buffer, pH 8.3, and 10-μl bacterial alkaline phosphatase (Sigma) were added to the cup and incubated at 37°C for 2 h. The Eppendorf cup was centrifuged at 13 000 g and the nucleoside-containing supernatant was transferred to a fresh Eppendorf cup and stored at 4°C.

Sharma S., Langhendries J.L., Watzinger P., Kötter P., Entian K.D, & Lafontaine D.L. (2015). Yeast Kre33 and human NAT10 are conserved 18S rRNA cytosine acetyltransferases that modify tRNAs assisted by the adaptor Tan1/THUMPD1. Nucleic Acids Research, 43(4), 2242-2258.

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Purification and Analysis of Biomolecules

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All chemicals and reagents were obtained at the highest purity available and were used without further purification unless stated. Benzonase, bacterial alkaline phosphatase, butylated hydroxytoluene, Pentostation (Deoxycoformycin), theophylline, acetonitrile and buffer salts were purchased from Sigma-Aldrich (Steinheim, Germany). Snake venom phosphodiesterase I was purchased from VWR (Darmstadt, Germany). Tetrahydrouridine was purchased from Merck (Darmstadt, Germany). Water purified through a Milli-Q system was used throughout our studies.

Schäck M.A., Jablonski K.P., Gräf S., Klassen R., Schaffrath R., Kellner S, & Hammann C. (2020). Eukaryotic life without tQCUG: the role of Elongator-dependent tRNA modifications in Dictyostelium discoideum. Nucleic Acids Research, 48(14), 7899-7913.

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Quantification of Yeast 25S rRNA Modifications

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RP-HPLC analysis as well as the mung bean nuclease protection assay were performed as described previously^{31 (link)}. Synthetic deoxyoligonucleotides complementary to the specific sequence of C633–G680 of yeast 25S rRNA were used for protection by hybridization to the rRNA. After digestion by mung bean nuclease (MBN Kit: M0250S, NEB) and 0.05 mg/ml RNase A (Sigma-Aldrich) purification of protected fragments from 8 M urea-PAGE (13%) was carried out by passive elution with 0.3 M NaAc by rotation at 4 °C overnight. Precipitation of eluted rRNA fragments was done using 100% EtOH.
Isolated fragments were digested with nuclease P1 and bacterial alkaline phosphatase (Sigma Aldrich) and subsequently the nucleosides were analyzed by RP-HPLC on a Supelcosil LC-18-S HPLC column (25 cm × 4.6 cm × 5 µm) equipped with a precolumn (4.6 cm × 20 mm) at 30 °C on an Agilent 1200 HPLC system.

Sharma S., Hartmann J.D., Watzinger P., Klepper A., Peifer C., Kötter P., Lafontaine D.L, & Entian K.D. (2018). A single N1-methyladenosine on the large ribosomal subunit rRNA impacts locally its structure and the translation of key metabolic enzymes. Scientific Reports, 8, 11904.

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Quantification of Nucleoside Modifications

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Total RNA was extracted from tissue with Trizol and precipitated with isopropyl alcohol according to the manufacturer’s instructions. Total tRNA was isolated using Nucleobond RNA/DNA 80 columns (Macherey-Nagel, Düren, Germany) and precipitated with isopropyl alcohol. Total tRNA was digested to nucleosides using nuclease P1 (Sigma) and bacterial alkaline phosphatase (Sigma) and analyzed as described earlier (42 (link)). t6A was used as internal control to normalize mcm⁵s²U quantification.

Bento-Abreu A., Jager G., Swinnen B., Rué L., Hendrickx S., Jones A., Staats K.A., Taes I., Eykens C., Nonneman A., Nuyts R., Timmers M., Silva L., Chariot A., Nguyen L., Ravits J., Lemmens R., Cabooter D., Van Den Bosch L., Van Damme P., Al-Chalabi A., Bystrom A, & Robberecht W. (2018). Elongator subunit 3 (ELP3) modifies ALS through tRNA modification. Human Molecular Genetics, 27(7), 1276-1289.

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Cloning and Mutagenesis of siRNA-Resistant DDX6

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p3xFlag-DDX6 was created using standard molecular biology techniques. Specifically DDX6 was PCR amplified from pmRFP-DDX6 (a generous gift from Nancy Kedersha, Brigham and Women’s Hospital) and cloned into pCR2.1-Topo. DDX6 was subcloned from pCR2.1-Topo into p3xFlag-CMV 7.1 (Sigma) using the HindIII and XbaI restriction enzyme sites. We then used site-directed mutagenesis with the QuikChange XLII to create an siRNA-resistant gene of DDX6 (3xFlag-RCKΔsi). In particular six nucleotides in the siRNA binding were mutated, while maintaining the correct amino acid sequence (from gcagaaaccctatgagatt to tcagaagccttacgaaatc). These mutations were introduced with the following primers: prCTP34 (5′-CTTTCCCTCTTAGTGTACAGAAGTTCATGAATTCCCATCTTCAGAAGCCTTACGAAATCAACCTGATGGAGGAACTAAC-3′) and prCTP35 (5′-GTTAGTTCCTCCATCAGGTTGATTTCGTAAGGCTTCTGAAGATGGGAATTCATGAACTTCTGTACACTAAGAGGGAAAG-3′). p3xFlag-BAP (Bacterial Alkaline Phosphatase, Sigma) was used as a control plasmid.

Biegel J.M., Henderson E., Cox E.M., Bonenfant G., Netzband R., Kahn S., Eager R, & Pager C.T. (2017). Cellular DEAD-box RNA helicase DDX6 modulates interaction of miR-122 with the 5′ untranslated region of hepatitis C virus RNA. Virology, 507, 231-241.

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Quantifying Oxidative DNA Damage in MWCNT-Treated Cells

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The levels of 8-oxodG in MNCNT-treated cells were measured by previously described method with modification [39 (link)]. A549 cells (5 × 10⁵ cells/ml) were treated with 1 μg/ml of MWCNT for indicated durations at 37 °C in DMEM containing 5 % (v/v) FBS and 100 mg/l kanamycin. The cells were lysed and treated with Proteinase K (Roche, Mannheim, Germany) for 1 h at 37 °C, and then sodium iodide was added. DNA was extracted and digested with nuclease P1 (Wako) and bacterial alkaline phosphatase (Sigma-Aldrich) to deoxynucleosides, and analyzed using HPLC coupled with an ECD (Coulochem II 5200A, ESA Biosciences, Chelmsford, MA, USA). The molar ratio of 8-oxodG to 2’-deoxyguanosine was measured based on ECD peak height of authentic 8-oxodG and the UV absorbance of 2’-deoxyguanosine at 254 nm.

Hiraku Y., Guo F., Ma N., Yamada T., Wang S., Kawanishi S, & Murata M. (2016). Multi-walled carbon nanotube induces nitrative DNA damage in human lung epithelial cells via HMGB1-RAGE interaction and Toll-like receptor 9 activation. Particle and Fibre Toxicology, 13, 16.

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