Fastap buffer

Manufactured by Thermo Fisher Scientific

FastAP buffer is a versatile buffer solution used in molecular biology applications. It is designed to facilitate the dephosphorylation of DNA and RNA fragments, a common step in various cloning and genetic engineering procedures. The buffer contains alkaline phosphatase, which catalyzes the removal of 5' phosphate groups from nucleic acids, enabling subsequent ligation or other manipulations. FastAP buffer is formulated to maintain optimal enzyme activity and stability, providing a convenient and efficient way to dephosphorylate DNA and RNA samples.

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Lab products found in correlation

0.22 μm filter, by Merck Group (3 mentions) Nuclease p1, by Fujifilm (3 mentions) C18 reverse phase column, by Agilent Technologies (2 mentions) Agilent 6460, by AB Sciex (2 mentions) Sciex 6500 triple quad mass spectrometer, by AB Sciex (2 mentions) Fastap thermosensitive alkaline phosphatase, by Thermo Fisher Scientific (2 mentions) Tegasept solution, by Merck Group (1 mentions) Tri reagent, by Merck Group (1 mentions) Eclipse xdb c18 column, by Agilent Technologies (1 mentions) Dynabeads mrna purification kit, by Thermo Fisher Scientific (1 mentions) C18 column, by Agilent Technologies (1 mentions) Minicomplete protease inhibitors, by Roche (1 mentions) Fastap, by Thermo Fisher Scientific (1 mentions) Fastap enzyme, by Thermo Fisher Scientific (1 mentions)

7 protocols using fastap buffer

Downregulation of beaf-32 in Drosophila

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To down regulate beaf-32, we generated flies expressing a UAS–RNAi transgene (BDSC number 35642) under the control of the actin5C-gal4 promoter (Stock #25374, Bloomington Stock Center, Indiana, USA). We utilized actin5C-gal4 flies as control. Flies were reared at 25°C on a standard diet (yeast: 38 g/l, yellow corn mill: 91 g/l, agar: 10 g/l, molasses: 8.7% v/v, propanoic acid (BioLab): 0.9% v/v, Tegasept solution (Sigma-Aldrich; 300 g/l in EtOH (BioLab)): 0.8% v/v)). After eclosion flies were kept in 12:12 light:dark cycles at 25°C. Newborn flies were entrained for 3 days in 12:12 light:dark conditions at different temperatures (18, 29°C). RNA was extracted from their heads using TRI Reagent, Sigma Aldrich. (Dnase treated) RNA was fragmented in the presence of 10× FastAP buffer (ThermoFisher Scientific) at 94°C for 3 min and placed on ice. RNA was then dephosphorylated with FastAP, cleaned (2× SPRI) and ligated to linker 1. Library preparation was continued as described above, starting from linker 1 3΄ ligation (for Exo-seq).

Afik S., Bartok O., Artyomov M.N., Shishkin A.A., Kadri S., Hanan M., Zhu X., Garber M, & Kadener S. (2017). Defining the 5΄ and 3΄ landscape of the Drosophila transcriptome with Exo-seq and RNaseH-seq. Nucleic Acids Research, 45(11), e95.

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Quantification of N6-methyladenosine

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RNA oligonucleotides or mRNAs were digested into nucleosides, and the amount of am⁶A was measured by using Agilent 6460 Triple Quad MS–MS with a 1290 UHPLC supplied with a ZORBAX Eclipse XDB-C18 column (UHPLC–QQQ–MS/MS) and calculated based on the standard curve generated by pure standards. For each sample, RNA was digested by using 1 U of nuclease P1 (Wako) in a 25-μl reaction containing 10 mM ammonium acetate at 37 °C for 16 h. Then, 1 μl of FastAP thermosensitive alkaline phosphatase and 3 μl of 10× FastAP buffer (Thermo Scientific) was added, and the reaction was incubated at 37 °C for 2 h. Samples were then filtered using a 0.22-μm filter (Millipore) and injected into LC–MS/MS. The nucleosides were quantified by using the nucleoside-to-base ion mass transitions of 282 to 150 (m⁶A), 323 to 191 (am⁶A), 268 to 136 (A) and 284 to 152 (G). Quantification was performed in comparison to the standard curve obtained from pure nucleoside standards run on the same batch of samples. The ratio of m⁶A to G was calculated based on the calibrated concentrations.

Hu L., Liu S., Peng Y., Ge R., Su R., Senevirathne C., Harada B.T., Dai Q., Wei J., Zhang L., Hao Z., Luo L., Wang H., Wang Y., Luo M., Chen M., Chen J, & He C. (2022). m6A RNA modifications are measured at single-base resolution across the mammalian transcriptome. Nature biotechnology, 40(8), 1210-1219.

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Quantifying RNA Modifications by UHPLC-MS

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50–200 ng of RNA were first digested by nuclease P1 (Wako, 1U) in 26 μL 1x P1 digestion buffer containing 25 mM NaCl, 2.5 mM ZnCl₂ at 42 °C for 2 hours. Next, 1 μL FastAP Thermosensitive Alkaline Phosphatase (ThermoFisher, 1 U/μL) and 3 μL 10x FastAP buffer (ThermoFisher) were added to the reaction and incubated at 37 °C for 2 hours. Reactions were then diluted into 60 μL and filtered by a 0.22 μm filter (Millipore). Filtered samples were injected into a C18 reverse phase column (Agilent) on a UHPLC coupled to an Agilent 6460 or a SCIEX 6500+ Triple Quad Mass Spectrometer in positive electrospray ionization mode. Quantitation was performed based on peak areas of characteristic nucleoside-to-base transitions, including 268-to-136 for A, 284-to-152 for G, 282-to-150 for m¹A and m⁶A with retention time at 0.8 minutes and 2.8 minutes, respectively.

Zhou H., Rauch S., Dai Q., Cui X., Zhang Z., Nachtergaele S., Sepich C., He C, & Dickinson B.C. (2019). Evolution of a Reverse Transcriptase to Map N1-Methyladenosine in Human mRNA. Nature methods, 16(12), 1281-1288.

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Quantifying RNA Modifications by UHPLC-MS

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RNA Methylation Quantification Protocol

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Total RNA was purified with TRIZOL reagent then subjected to two rounds of poly(A) selection with the Dynabeads mRNA purification kit (Invitrogen). 25 ng of the purified mRNA was digested by nuclease P1 (1 U) in 20 μl of buffer containing 20 mM NH4OAc pH 5.5 at 42 °C for 2 h, followed by the addition FastAP buffer (2.3μL, Thermo Scientific) and alkaline phosphatase (1 U) and incubation at 37 °C for 4 h. The sample was then filtered (0.22 μm pore size, 4 mm diameter, Millipore), and 5 μl of the solution was injected into the LC-MS/MS. The nucleosides were separated by reverse-phase ultra-performance liquid chromatography on a C18 column (Agilent) with online mass spectrometry detection using a Sciex 6500+ triple-quadrupole LC mass spectrometer in positive electrospray ionization mode. The nucleosides were quantified by using the nucleoside-to-base ion mass transitions of 282 to 150 (m⁶A) and 268 to 136 (A). Quantification was carried out by comparison with a standard curve obtained from pure nucleoside standards run with the same batch of samples. The ratio of m⁶A to A was calculated based on the calibrated concentrations.

Wei J., Harada B.T., Lu D., Ma R., Gao B., Xu Y., Montauti E., Mani N., Chaudhuri S., Gregory S., Weinberg S., Zhang D., Green R., He C, & Fang D. (2021). HRD1-mediated METTL14 degradation regulates m6A mRNA modification to suppress ER proteotoxic liver disease. Molecular cell, 81(24), 5052-5065.e6.

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Phosphorylation Analysis Using Phos-tag Gel

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Shisa9/CKAMP44 phosphorylation was assessed using the Phos‐tag system (Wako, Neuss, Germany), which facilitates phosphorylation‐dependent retarded protein migration through a polyacrylamide gel, and subsequent WB. Phos‐tag gel electrophoresis (6–8% polyacrylamide supplemented with 50 μm Phos‐tag) was established using an optimised protocol with Zn²⁺ in a bis/tris‐buffered neutral pH gel system either as described previously 48 or according to the manufacturer's recommendations. Phos‐tag gels were generally blotted using a wet transfer blotting set‐up. As a control for phosphorylated proteins, the proteins were dephosphorylated using a thermosensitive alkaline phosphatase (FastAP; Thermo Fisher Scientific). The transfected cells were lysed in 1× FastAP buffer (Thermo Fisher Scientific) containing 1% Triton‐X and Mini Complete protease inhibitors without EDTA (Roche). The lysates were cleared by centrifugation and proteins were subsequently dephosphorylated for 2 h at 37 °C using FastAP.

Kunde S., Rademacher N., Zieger H, & Shoichet S.A. (2017). Protein kinase C regulates AMPA receptor auxiliary protein Shisa9/CKAMP44 through interactions with neuronal scaffold PICK1. FEBS Open Bio, 7(9), 1234-1245.

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Ipl1-Spc105 Kinase Assay and Phosphatase Treatment

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The in vitro kinase assay was performed using the Sli15(INBOX)-Ipl1-6His (construct courtesy of the Biggins lab), which was purified using the methods outlined above with the HEPES purification buffer. The Sli15(INBOX)-Ipl1-6His was preactivated by incubating with 0.2 mM ATP-MgCl₂ (pH 7.0) and 1× phosphatase inhibitor cocktail (PhoSTOP-Roche) for 20 min at 30°C. The activated Sli15(INBOX)-Ipl1-6His was then incubated with MBP-Spc105(10xMEED)-TST in HEPES kinase buffer (20 mM HEPES, pH 7.4, 100 mM KCl, 10 mM MgCl₂, 1 mM DTT) + 1× PhoSTOP Roche for 20 min at 30°C.
For phosphatase treatment, the Ipl1 + Spc105 kinase mixture was bound to washed magnetic Strep-Tactin-XT beads for 30 min at room temperature. The beads were next resuspended in 1× FastAP buffer (Thermo Fisher Scientific) with or without 10 U of FastAP enzyme (Thermo Fisher Scientific) and incubated for 1 h at 37°C. After the beads were washed three times with 1× FastAP buffer, the mixture was eluted into 1× SDS and run on a 10% SDS–PAGE gel. The proteins were detected via Western blotting and/or Coomassie staining.

Audett M.R., Johnson E.L., McGory J.M., Barcelos D.M., Szalai E.O., Przewloka M.R, & Maresca T.J. (2022). The microtubule- and PP1-binding activities of Drosophila melanogaster Spc105 control the kinetics of SAC satisfaction. Molecular Biology of the Cell, 33(1), ar1.

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