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Bacterial alkaline phosphatase

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Bacterial alkaline phosphatase is an enzyme commonly used in molecular biology and biochemistry applications. It catalyzes the removal of phosphate groups from various substrates, including nucleic acids and proteins. The enzyme is derived from bacterial sources and exhibits optimal activity under alkaline conditions.

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Lab products found in correlation

10k mwco columns, by Thermo Fisher Scientific (2 mentions) G25 spin column, by GE Healthcare (2 mentions) Nuclease p1, by Merck Group (2 mentions) Rnase t1, by Thermo Fisher Scientific (1 mentions) Benzonase nuclease, by Merck Group (1 mentions) Hypersil gold aq column, by Thermo Fisher Scientific (1 mentions) Agilent 6470, by Agilent Technologies (1 mentions) Nanodrop 2000c, by Thermo Fisher Scientific (1 mentions) Sodium acetate, by Merck Group (1 mentions) Nuclease p1, by New England Biolabs (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Zinc chloride, by Merck Group (1 mentions) Phosphodiesterase 1, by Merck Group (1 mentions) 6490 triple quadrupole mass spectrometer, by Agilent Technologies (1 mentions) Hypersil gold aq reverse phase column, by Thermo Fisher Scientific (1 mentions) Lc 20ad, by Shimadzu (1 mentions) 1260 infinity hplc, by Agilent Technologies (1 mentions)

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Alkaline phosphatase (63 citations)

8 protocols using bacterial alkaline phosphatase

1

Enzymatic Methylation and Analysis of tRNA

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The 80 μl MTase reaction mixtures consisting of 10 μg T7 in vitro tRNA transcript, 5 μM recombinant PaTrmB protein, 50 mM Tris–HCl (pH 8.0), 5 mM MgCl2, 50 mM KCl and 50 μM SAM (Sigma) were assembled and incubated at 37°C for 1 h. The m7G-modified tRNA was subsequently purified from the MTase reaction mixtures by filtration using 10K MWCO columns (Ambion). Five micrograms of methylated tRNA was digested into fragments with RNase T1 or RNase U (Invitrogen) and then dephosphorylated with bacterial alkaline phosphatase (Invitrogen) at 37°C for 4 h in the presence of deaminase inhibitors and antioxidants. To purify the digested fragments from the solution, 100 μl of the digestion reaction was applied to Strata™ solid-phase extraction columns (Phenomenex). The purified fragments were then fractionated on TSK-gel Amide-80 column (2.0 mm ID × 150 mm, 3 μm particle size) with a gradient made of solvent A (8 mM ammonium acetate) and solvent B (100% acetonitrile) before sequencing using a quadrupole time-of-flight mass spectrometer (Agilent 6520) operated in negative ion mode as previously described (27 (link)).
Thongdee N., Jaroensuk J., Atichartpongkul S., Chittrakanwong J., Chooyoung K., Srimahaeak T., Chaiyen P., Vattanaviboon P., Mongkolsuk S, & Fuangthong M. (2019). TrmB, a tRNA m7G46 methyltransferase, plays a role in hydrogen peroxide resistance and positively modulates the translation of katA and katB mRNAs in Pseudomonas aeruginosa. Nucleic Acids Research, 47(17), 9271-9281.
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2

Formaldehyde Fixation and Immunostaining

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Neurons were fixed with 4% formaldehyde in PBS for 25 min at room temperature and then washed 3 times with PBS. The cells were subsequently permeabilized with 0.2% Triton X-100 for 5 min at room temperature and blocked with a solution containing 5% normal goat serum in PBS for 1 h. The cells were then incubated with the appropriate primary antibodies in PBS over night at 4 °C, after which they were incubated with the appropriate secondary antibodies in PBS containing 5% normal goat serum for 1 h. For alkaline phosphatase treatment of the fixed neurons, we incubated the samples with 100 µl of alkaline phosphatase solution containing 450 U of bacterial alkaline phosphatase (Invitrogen), 5% β-mercaptoethanol, and 10 mM Tris-HCl (pH 8.0) for 3 h at 32 °C.
Cornelia Koeberle S., Tanaka S., Kuriu T., Iwasaki H., Koeberle A., Schulz A., Helbing D.L., Yamagata Y., Morrison H, & Okabe S. (2017). Developmental stage-dependent regulation of spine formation by calcium-calmodulin-dependent protein kinase IIα and Rap1. Scientific Reports, 7, 13409.
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3

Comprehensive RNA Modification Analysis

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Five micrograms of total tRNA was treated with Benzonase nuclease (Sigma), bacterial alkaline phosphatase (Invitrogen), and phosphodiesterase in the presence of deaminase inhibitors (0.5 μg/ml coformycin and 5 μg/ml tetrahydrouridine) and antioxidants (50 μM desferrioxamine and 50 μM butylatedhydroxytoluene) at 37°C overnight to generate ribonucleoside products. Proteins were removed from the digested product by filtration with 10K MWCO columns (Ambion). The ribonucleosides were fractionated by using a reversed-phase column (Thermo Hypersil Gold aQ column) with a gradient made of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) prior to introduction into an electrospray ionization triple quadrupole mass spectrometer (Agilent 6470), which was operated in the positive ion mode as described previously (27 (link),28 (link)). The modified ribonucleosides were identified by their HPLC retention times, CID fragmentation patterns, fragmentor voltages, and collision energies in comparison with available chemical synthetic standards. The level of each modified ribonucleoside was quantified from the MRM signal intensity and normalized by dividing by the summed signals of adenosine, guanosine, cytidine and uridine from the sample.
Thongdee N., Jaroensuk J., Atichartpongkul S., Chittrakanwong J., Chooyoung K., Srimahaeak T., Chaiyen P., Vattanaviboon P., Mongkolsuk S, & Fuangthong M. (2019). TrmB, a tRNA m7G46 methyltransferase, plays a role in hydrogen peroxide resistance and positively modulates the translation of katA and katB mRNAs in Pseudomonas aeruginosa. Nucleic Acids Research, 47(17), 9271-9281.
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4

Quantification of Modified Nucleosides in P. xylostella

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Total RNA for high‐performance liquid chromatography coupled to tandem mass spectrometry (HPLC‐MS/MS) assays were isolated from 30 midguts of fourth‐instar P. xylostella larvae of the DBM1Ac‐S, Cry1Ac‐treated DBM1Ac‐S, and NIL‐R strains using the TRIzol reagent (Invitrogen). The concentrations of the RNA samples were determined using a Nanodrop 2000c (Thermo Fisher Scientific). Single nucleosides were generated from total RNA by a digestion buffer containing phosphodiesterase I (0.01 U) (Sigma Aldrich), nuclease P1 (1 U) (New England Biolabs), 2.5 mM zinc chloride (Sigma Aldrich) and 25 mM sodium acetate (Sigma Aldrich) at pH 6.8 for 2.5 h at 37 °C, followed by dephosphorylation with bacterial alkaline phosphatase (10 U) (Invitrogen) for 1 h at 37 °C. Nucleosides were separated on a Hypersil GOLD aQ reverse phase column (Thermo Scientific) and quantified with the nucleoside‐to‐base ion mass transitions of 258.1–126.1 for m1A and m5C, and 282.1–150.1 for m6A using HPLC‐MS/MS analysis on an Agilent 6490 Triple Quadrupole mass spectrometer.
Guo Z., Bai Y., Zhang X., Guo L., Zhu L., Sun D., Sun K., Xu X., Yang X., Xie W., Wang S., Wu Q., Crickmore N., Zhou X, & Zhang Y. (2023). RNA m6A Methylation Suppresses Insect Juvenile Hormone Degradation to Minimize Fitness Costs in Response to A Pathogenic Attack. Advanced Science, 11(6), 2307650.
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5

Radioactive Labeling of RNA Aptamers

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RNA aptamers were radioactively labeled by 32P at the 5′-end, initially by removing the 5′-terminal phosphate group with bacterial alkaline phosphatase (Life Technologies) at 65 °C for 1 hr and then purifying them by phenol/chloroform extraction followed by ethanol precipitation. 3 pmol of each dephosphorylated aptamer was incubated with 32P-labeled γ-ATP and T4-polynucleotide kinase (NEB) at 37 °C for 45-minute. Radiolabeled aptamers were finally purified using G25-spin column (GE Healthcare) following the manufacturer’s instructions. Incorporated radioactivity was quantified using a scintillation isotope counter.
Kahsai A.W., Wisler J.W., Lee J., Ahn S., Cahill TJ I.I.I., Dennison S.M., Staus D.P., Thomsen A.R., Anasti K.M., Pani B., Wingler L.M., Desai H., Bompiani K.M., Strachan R.T., Qin X., Alam S.M., Sullenger B.A, & Lefkowitz R.J. (2016). Conformationally Selective RNA Aptamers Allosterically Modulate the β2-Adrenoceptor. Nature chemical biology, 12(9), 709-716.
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6

Radioactive Labeling of RNA Aptamers

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RNA aptamers were radioactively labeled by 32P at the 5′-end, initially by removing the 5′-terminal phosphate group with bacterial alkaline phosphatase (Life Technologies) at 65 °C for 1 hr and then purifying them by phenol/chloroform extraction followed by ethanol precipitation. 3 pmol of each dephosphorylated aptamer was incubated with 32P-labeled γ-ATP and T4-polynucleotide kinase (NEB) at 37 °C for 45-minute. Radiolabeled aptamers were finally purified using G25-spin column (GE Healthcare) following the manufacturer’s instructions. Incorporated radioactivity was quantified using a scintillation isotope counter.
Kahsai A.W., Wisler J.W., Lee J., Ahn S., Cahill TJ I.I.I., Dennison S.M., Staus D.P., Thomsen A.R., Anasti K.M., Pani B., Wingler L.M., Desai H., Bompiani K.M., Strachan R.T., Qin X., Alam S.M., Sullenger B.A, & Lefkowitz R.J. (2016). Conformationally Selective RNA Aptamers Allosterically Modulate the β2-Adrenoceptor. Nature chemical biology, 12(9), 709-716.
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7

Mass Spectrometry Analysis of Modified mRNA Nucleosides

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Mass spectrometry analysis of C, ac4C, and 5fC incorporation into IVT mRNAs was assessed following nuclease digestion. Briefly, in vitro transcribed mRNAs were treated with nuclease P1 (1U/10 μg RNA, #N8630, Sigma) in 50 μL of buffer containing 100 mM ammonium acetate [pH 5.5] for 16 h at 37 °C. Samples were heated at 60 °C for 5 minutes, returned to 37 °C for an additional hour and 5 μL of 1 M ammonium bicarbonate [pH 8.3] and Bacterial Alkaline Phosphatase (0.5U/10 μg RNA, #18011–015, ThermoFisher) were added. Samples were further incubated at 37 °C for 2 h, adjusted to 150 μL with nuclease-free water and filtered to remove enzymatic constituents (Amicon Ultra 3K, #UFC500396). Following lyophilization, samples were reconstituted in 10 μL RNase-free water and analyzed via LC-MS/MS using a reverse phase chromatography (Shimadzu LC-20AD) coupled to a triple-quadrupole mass spectrometer (Thermo TSQ-ultra) operated in positive electrospray ionization mode. Quantification was accomplished by monitoring nucleoside-to-base ion transitions and generating standard curves for each nucleoside using the stable isotope dilution internal standardization method. (Fig. S1)
Nance K.D., Gamage S.T., Alam M.M., Yang A., Levy M.J., Link C.N., Florens L., Washburn M.P., Gu S., Oppenheim J.J, & Meier J.L. (2021). Cytidine acetylation yields a hypoinflammatory synthetic messenger RNA. Cell chemical biology, 29(2), 312-320.e7.
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8

HPLC Detection of N4-Acetylcytidine (ac4C)

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Detection of ac4C by HPLC was performed as described previously (Sinclair et al., 2017 (link)). Briefly, RNA was incubated with 1U/10 μg RNA of nuclease P1 (Sigma-Aldrich) in 100 mM ammonium acetate [pH 5.5] for 16 hr at 37 °C. Five microliter of 1 M ammonium bicarbonate [pH 8.3] and 0.5U/10 μg RNA of Bacterial Alkaline Phosphatase (ThermoFisher Scientific) were added for 2 hrs at 37 °C. Following digestion, samples were lyophilized and reconstituted in 10 μL RNase-free water and injected into an Agilent Technologies 1260 Infinity HPLC equipped with a UV detector (Agilent Technologies). Nucleosides were separated on a Kinetex 2.6u C18 100A 100×2.1 mm column at a flow rate of 0.25 mL/min. UV detector was set at 254 nm with a band width of 4 nm. Buffer A: 0.01% formic acid; buffer B: 50% acetonitrile, 0.01% formic acid [pH 3.5] with the gradient as follows: 0−1 min, 100% A; 1−2.4 min, 99.8% A; 2.4−3.8 min, 99.2% A; 3.8− 5.2 min, 98.2% A; 5.2−6.6 min, 96.8% A; 6.6−10 min, 95% A; 10− 12.5 min, 92% A; 12.5−18 min, 70% A; 18−18.5 min, 0% A; 18.5−20 min, 0% A; 20−21 min, 100% A; 21−30 min, 100% A.
Arango D., Sturgill D.M., Alhusaini N., Dillman A.A., Sweet T.J., Hanson G., Hosogane M., Sinclair W.R., Nanan K.K., Mandler M.D., Fox S.D., Zengeya T.T., Andresson T., Meier J.L., Coller J, & Oberdoerffer S. (2018). Acetylation of cytidine in messenger RNA promotes translation efficiency. Cell, 175(7), 1872-1886.e24.
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