RNA samples were extracted as previously shown. Add 1 μg of RNA samples into the buffer solution, completely enzymolysis the samples into nucleosides at 37 °C under the action of phosphodiesterase (0.002 U/μL; Sigma-Aldrich, P3243), S1 nuclease (180 U/μL; Takara, 2410A) and alkaline phosphatase (30 U/μL; Takara, 2250A), and re extract the enzymatically hydrolyzed sample with chloroform-method. Put the obtained upper aqueous solution into the injection bottle and analyze it via LC-MS/MS. The chromatographic column mainly adopts Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm, 100 mm × 2.1 mm i.d.). At 40 °C, the column flow rate was set to 0.3 mL/min. For mass spectrum, the temperature of the electrospray ion source was set to 550 °C, and the mass spectrum voltage is set to 5 500 V under positive electrospray ionization mode.
Phosphodiesterase
Manufactured by Merck Group
Sourced in United States
Phosphodiesterase is a laboratory equipment used to measure the enzymatic activity of phosphodiesterase, which is an enzyme that catalyzes the hydrolysis of cyclic nucleotides such as cAMP and cGMP. This equipment is commonly used in biochemical research and drug discovery applications to study the role of phosphodiesterase in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Alkaline phosphatase, by Merck Group (6 mentions)
Genejet genomic dna purification kit, by Thermo Fisher Scientific (3 mentions)
Nuclease p1, by Merck Group (3 mentions)
Acquity uplc hss t3 c18 column, by Waters Corporation (2 mentions)
S1 nuclease, by Takara Bio (2 mentions)
Dna methylation elisa kit, by Cayman Chemical (2 mentions)
Alkaline phosphatase, by Roche (1 mentions)
Nanodrop machine, by Thermo Fisher Scientific (1 mentions)
5 aza 2 deoxycytidine 15n4, by LGC (1 mentions)
Savant speed vac plus sc210a, by Thermo Fisher Scientific (1 mentions)
Benzonase, by Merck Group (1 mentions)
Milli q water, by Merck Group (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Alkaline phosphatase, by Takara Bio (1 mentions)
Qtrap 6500, by AB Sciex (1 mentions)
Chloroform, by Sinopharm (1 mentions)
9 protocols using phosphodiesterase
1
Quantitative Analysis of RNA Nucleosides
2
DNA Methylation Quantification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was extracted from samples using the GeneJET Genomic DNA Purification Kit (Thermo Fisher, Suwanee, GA) following manufacturer's instructions. Thereafter, the isolated DNA was treated with nuclease P1, alkaline phosphatase, and phosphodiesterase (Sigma-Aldrich, St. Louis, MO) (27 (link)). CpG methylation of the samples was quantified with a DNA methylation ELISA kit (Cayman Chemical, Ann Arbor, Michigan) following manufacturer's instructions.
3
Purification and labeling of Rho proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All proteins were purified according to the protocols described (3 (link), 71 (link), 72 (link)). Briefly, Escherichia coli strains pLysS BL21(DE3), CodonPlusRIL, or BL21(Rosetta) were transformed and used to purify the respective protein. Protein extraction was carried out by incubating cells at 4 °C with DNase I (10 μg ml−1) and lysozyme (10 μg ml−1) followed by cell lysis using a microfluidizer (model M110S, Microfluidics Corp.). Bacterial lysates were centrifuged to collect soluble fractions, and tagged proteins were isolated from the supernatant via Ni-NTA or GST affinity purification. If needed, the GST tag was cleaved with tobacco etch virus protease (4 units mg−1, 4 °C, overnight), and proteins were subjected to gel filtration using a standard buffer containing 30 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm MgCl2, and 3 mm DTT. All purified proteins were analyzed by SDS-PAGE (Fig. 1 ) and stored as either tag-fused or cleaved protein at −80 °C.
Nucleotide-free RHO proteins were prepared using alkaline phosphatase (Roche Applied Science) and phosphodiesterase (Sigma) at 4 °C as described (73 (link)). Various fluorescence reporter groups, including mant and tamra, have been coupled to 2′ (3′)-hydroxyl group of the ribose moiety of GDP and GppNHp via ethylenediamine (EDA) to obtain fluorescent nucleotide (Jena Bioscience).
Nucleotide-free RHO proteins were prepared using alkaline phosphatase (Roche Applied Science) and phosphodiesterase (Sigma) at 4 °C as described (73 (link)). Various fluorescence reporter groups, including mant and tamra, have been coupled to 2′ (3′)-hydroxyl group of the ribose moiety of GDP and GppNHp via ethylenediamine (EDA) to obtain fluorescent nucleotide (Jena Bioscience).
4
DNA Methylation Quantification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was extracted from samples using the GeneJET Genomic DNA Purification Kit (Thermo Fisher, Suwanee, GA, USA) following the manufacturer’s instructions. DNA quantity and quality was determined by a Nanodrop machine (Thermo Fisher). An A260/280 ratio over 1.8 was considered acceptable. Thereafter, the isolated DNA was treated with nuclease P1, alkaline phosphatase, and phosphodiesterase (Sigma-Aldrich, St. Louis, MO, USA) [36 (link)]. CpG methylation of the samples was quantified with a DNA methylation ELISA kit (Cayman Chemical, Ann Arbor, MI, USA) following the manufacturer’s instructions.
5
Quantitative DNA/RNA Modification Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For duplicate measurement, 1–10 μg of extracted DNA, together with 5 μl of 50 μM 5-aza-2′-deoxycytidine-15N4 (internal standard, Toronto Research Chemicals, Toronto, ON, Canada), was added to 10 μl of 20 mg/ml NaBH4 solution. In case of RNA, 1–10 μg of extracted RNA, together with 5 μl of 50 μM 5-azacytidine-15N4 (internal standard, Thermo Fisher Scientific), was added to 10 μl of 20 mg/ml NaBH4 solution. The mixture was incubated at room temperature with agitation for 20 min, and neutralised with 1 μl 5 M HCl. Reduced DNA or RNA were digested with 1 h of incubation at 37 °C following the addition of 40 μl of Digest Mix, an aqueous solution containing 50 U/ml benzonase (Sigma Aldrich, St Louis, MO, USA), 60 mU/ml phosphodiesterase (Sigma Aldrich), 20 U/ml alkaline phosphatase (Sigma Aldrich), 20 mM Tris HCl pH 8, 100 mM NaCl and 20 mM MgCl2 as previously described.38 (link) Samples were dried under vacuum (Savant Speedvac Plus SC210A, Thermo Fisher Scientific) and resuspended in 50 μl of CE buffer (10 mM Tris HCl pH 8.0 and 0.5 mM EDTA in Milli-Q water, Merck Millipore, Bedford, MA, USA) for LC–MS analysis.
6
DNA Methylation Quantification in Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was extracted from HepG2 and BeWo cells using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific) as per the manufacturer’s instructions. DNA was treated with nuclease P1, alkaline phosphatase, and phosphodiesterase (Sigma-Aldrich).19 (link) CpG methylation of the samples was quantified using a DNA methylation enzyme-linked immunosorbent assay (ELISA) kit (Cayman Chemical) as per the manufacturer’s instructions.
7
Global DNA Methylation Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Global DNA methylation was estimated using hydrolysis of genomic DNA followed by specific detection and quantification of the 5-methylcytosine content. First, DNA (5 μg) containing 0•1 mM deferoxamine was hydrolysed with Tris buffer including 200 mM HCl/MgCl 2 (pH 7•4) and DNAse 1 (Sigma Aldrich, EUA) for 1 h at 1000 rpm and 37°C. DNA was incubated with phosphodiesterase (0•0005 units) (Sigma Aldrich) and alkaline phosphatase (1•2 units) for 1 h at 37°C. After that, the final volume (60 μl) was centrifuged for 10 min at 3000 rpm and the supernatant was collected. Aliquots of hydrolysed DNA were injected into the HPLC with diode-array detection analytical system (Shimadzu Corporation). Last, the percentage of the global DNA methylation was estimated. Other additional information about the method was described in a different study (27) .
8
Purification and Characterization of RHO GTPases
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All proteins were produced using Escherichia coli and baculovirus-insect cell expression system as described [34 (link)]. Glutathione S-transferase (GST) fusion proteins were isolated by affinity chromatography on a glutathione Sepharose column in the first step and purified by size exclusion chromatography after proteolytic cleavage of GST in the second step [86 (link)]. His-tagged proteins were isolated from SF9 insect cells, using affinity chromatography on Ni-NTA columns. The quality of the proteins was analyzed by 12% SDS-PAGE. Protein concentrations were determined using Bradford reagent (Coomassie dye reagent; Sigma, (Steinheim, Germany)), and the GDP concentration in the case of purified RHO GTPases was determined using HPLC [87 ]. Nucleotide-free RHO proteins were prepared using alkaline phosphatase (Sigma Aldrich, Deisenhofen, Germany)and phosphodiesterase (Sigma Aldrich, Deisenhofen, Germany) at 4 °C as previously described [87 ]. RHO GTPases were loaded with 2-deoxy-3-O-N-methylanthraniloyl GDP (mdGDP); the fluorescent reporter group methyl-anthraniloyl (m) was attached to the 3′-OH group, and can, due to the lack 2′-OH, not isomerize between 2′- and 3-OH groups as compared to mGDP [88 (link)].
9
Comprehensive RNA Metabolite Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Buffer, S1 nuclease (Takara, Japan), phosphodiesterase (Sigma-Aldrich, USA), and alkaline phosphatase (Takara, Japan) were added into 1 µg of total RNA to completely digest RNA into nucleic acid at 37°C, then extracted with chloroform (Sinopharm Chemical Reagent co., Ltd. China) and took the upper layer water sample for subsequent LC-ESI-MS/MS analysis. The liquid phase conditions are as follows: Chromatographic column: Waters ACQUITY UPLC HSS T3 C18 column (1.8 µm, 100 mm × 2.1 mm i.d.); gradient elution program: 0 min A/B is 95:5 (V/V), 1.0 min A/B is 95:5 (V/V), 9.0 min A/B is 5:95 (V/V), 11.0 min A/B is 95:5 (V/V), 11.1 min A/B is 95:5 (V/V), 14.0 min A/B is 95:5 (V/V) (phase A is ultrapure water (2mM ammonium bicarbonate), phase B is methanol (2mM ammonium bicarbonate); flow rate was at 0.3 mL/min; column temperature is 40°C; the injection volume is 10 µL. Then the MS/MS analysis conditions are as follows: Electrospray Ionization (ESI) temperature is 550°C, mass spectrometer voltage is 5500v under the positive ion mode, and Curtain Gas (CUR) is 35 psi. In Q-Trap 6500+ (SCIEX, USA), each ion pair is scanned according to optimized Declustering Potential (DP) and Collision Energy (CE). Finally, build an MWDB (Metware Database) database based on the standard, and perform qualitative analysis on the data detected by mass spectrometry.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!