Detailed methods for biotinylating and purifying thio-tagged RNAs have been published9 (link),15 (link), and detailed protocols [AU correct as edited?] including the most recent improvements are available upon request. Relevant changes to previous protocols are summarized here. Biotinylation of RNA was performed using EZ-Link Biotin-HPDP (N-(6-(Biotinamido)hexyl)-3'-(2'-pyridyldithio)-propionamide; Pierce), as previously described. Biotinylation reactions contained 10 mM Tris (pH 7.4), 1 mM EDTA, and 2 μl of a 1 mg/ml Biotin-HPDP solution (in dimethylformamide) per 2 μg of RNA. The reaction volume was adjusted with water so that the concentration of Biotin-HPDP was equal to 30% of the final reaction volume. Biotinylation reactions were incubated in the dark for 3 hrs at 25°C prior to RNA precipitation. Biotinylated RNA was detected by blotting and probing with streptavidin-HRP as previously described. Purification of biotinylated TU-tagged RNA was performed as previously described with the following modifications: 2 μl of MPG streptavidin beads (PureBiotech) were used per μg of input RNA. The input RNA was always at a concentration of 0.5 μg/μl. After blocking with yeast tRNA and washing, beads were resuspended in the input RNA sample plus a volume of MPG buffer (1M NaCl, 10 mM EDTA, 100 mM Tris pH 7.4, in RNAse free H2O) equal to 1/3 the input RNA volume. Beads plus RNA were incubated at room temperature for 20 minutes prior to collecting the non-bound sample and performing washes with MPG buffer (one five minute wash at room temperature, two one minute washes at room temperature, a one minute wash in 65°C MPG buffer, and a final one minute wash at room temperature). After the removal of as much MPG buffer as possible, TU-tagged RNA was eluted by incubating the beads for ten minutes in freshly prepared 5% 2-mercaptoethanol. RNA was precipitated using isopropanol and linear acrylamide. After resuspending RNA in water, samples were placed in the magnetic stand again to remove any remaining MPG beads. For the purification procedures, input amounts of biotinylated RNA ranged from 14 μg to 20 μg.
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Propionamide
Propionamide
Propionamide is a synthetic organic compound with the chemical formula CH3CH2CONH2.
It is used in various research and industrial applications, including as an intermediate in the production of other chemicals.
Propionamide research is important for understanding its properties, potential uses, and optimizing production methods.
PubCompare.ai, an AI-driven platform, enhanes reproducibility and accuracy in Propionamide research by helping researchers locate protocols from literature, preprints, and patents, and leverage AI-driven comparisons to identify the best protocols and products.
This can help optimize Propionamide research and development effrts.
It is used in various research and industrial applications, including as an intermediate in the production of other chemicals.
Propionamide research is important for understanding its properties, potential uses, and optimizing production methods.
PubCompare.ai, an AI-driven platform, enhanes reproducibility and accuracy in Propionamide research by helping researchers locate protocols from literature, preprints, and patents, and leverage AI-driven comparisons to identify the best protocols and products.
This can help optimize Propionamide research and development effrts.
Most cited protocols related to «Propionamide»
2-Mercaptoethanol
Acrylamide
Biotin
Biotinylation
Buffers
Dimethylformamide
Edetic Acid
Endoribonucleases
Isopropyl Alcohol
N-(6-(biotinamido)hexyl)-3'-(2'-pyridyldithio)propionamide
propionamide
Saccharomyces cerevisiae
Sodium Chloride
Streptavidin
Transfer RNA
Tromethamine
2-Mercaptoethanol
Acrylamide
Biotin
Biotinylation
Buffers
Dimethylformamide
Edetic Acid
Endoribonucleases
Isopropyl Alcohol
N-(6-(biotinamido)hexyl)-3'-(2'-pyridyldithio)propionamide
propionamide
Saccharomyces cerevisiae
Sodium Chloride
Streptavidin
Transfer RNA
Tromethamine
The raw data were processed using ProteinLynx Global Server (PLGS, version 2.4 Waters Corporation, Milford, MA) software, as described previously 16 . The following parameters were used: background subtraction of polynomial order five adaptive with a threshold of 30%, two smoothings with a window of three channels in Savitzky‐Golay mode and centroid calculation of top 80% of peaks based on a minimum peak width of four channels at half height. The resulting pkl files were submitted for database search and protein identification to the in‐house Mascot server for database search (www.matrixscience.com , Matrix Science, London, UK, version 2.5.1) using the following parameters: databases from NCBI_20150706 database (selected for Rattus, 84,157 entries), parent mass error of 1.3 Da, product ion error of 0.8 Da, one 13C isotope, enzyme used: trypsin, one missed cleavage, propionamide as cysteine fixed modification and methionine oxidized as variable modification. To identify the false‐negative results, we used additional parameters such as different databases or organisms, a narrower error window for the parent mass error (1.2 and then 0.2 Da) and for the product ion error (0.6 Da), and up to two missed cleavage sites for trypsin. In addition, the pkl files were also searched against in‐house PLGS database version 2.4 (Waters Corporation, Milford, MA, USA) using searching parameters similar to the ones used for Mascot search. The Mascot and PLGS database search provided a list of proteins for each gel band. These data were then uploaded on the Scaffold version 4.2.1 software (Proteome Software Inc., Portland, OR, USA) for quantitative analysis.
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Acclimatization
Carbon-13
Cysteine
Cytokinesis
Enzymes
Methionine
Parent
propionamide
Proteins
Proteome
Rattus
Staphylococcal Protein A
Trypsin
Coomassie-stained gel spots corresponding to the same spots that showed statistically significant differential abundance in the 2D-DIGE gels were excised manually and were washed and digested according to previously described methods [53 (link)]. The mixture of tryptic peptides (0.5 µL) derived from each protein was spotted onto a MALDI target (384 Anchorchip MTP 800 µm Anchorchip; Bruker Daltonik, Bremen, Germany) together with 0.5 μL of matrix (10 mg α-cyano-4-hydroxycinnamic acid (CHCA) in 1 μL of 30% CH3CN and 0.1% aqueous CF3COOH) and then left to dry (room temperature, RT) before MS analysis. Spectra were acquired on a MALDI-TOF MS (UltraFlexTrem, Bruker Daltonics, Bremen, Germany) in the positive mode (target voltage 25 kV, pulsed ion extraction voltage 20 kV). The reflector voltage was set to 21 kV and the detector voltage to 17 kV. Peptide mass fingerprints (PMF) were calibrated against a standard (peptide calibration standard II, Bruker Daltonics). The PMFs were processed using Flex Analysis software (version 2.4, Bruker Daltonics). MS data were interpreted by using BioTools v3.2 (Bruker Daltonics), together with the Mascot search algorithm (version 2.0.04 updated 9 May 2015; Matrix Science Ltd., London, UK). Mascot parameters were as follows: fixed cysteine modification with propionamide, variable modification due to methionine oxidation, one missed cleavage site (i.e., in case of incomplete trypsin hydrolysis), and a mass tolerance of 100 ppm. Identified proteins were accepted as correct if they showed a Mascot score greater than 56 and p < 0.05. Not all the spots of interest could be identified because some proteins were of low abundance and did not yield sufficiently intense mass fingerprints; other spots were mixtures of multiple proteins.
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Coumaric Acids
Cysteine
Cytokinesis
Exanthema
Fingerprints, Peptide
Gels
Hydrolysis
Immune Tolerance
Methionine
Peptides
propionamide
Proteins
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Trypsin
Two-Dimensional Difference Gel Electrophoresis
Coomassie-stained gel spots were excised manually, washed, and digested according to previously described methods [49 (link)]. The mixture of tryptic peptides (0.5 μL) derived from each protein was spotted onto a MALDI target (384 anchorchip MTP 800 μm Anchorchip; Bruker Daltonik, Germany) together with 0.5 μL of matrix (10 mg α-cyano-4-hydroxycinnamic acid (CHCA) in 1 mL of 30% CH3CN and 0.1% aqueous CF3COOH) and left to dry (room temperature, RT) before MS analysis. Spectra were acquired on a MALDI-TOF MS (UltraFlexTrem, Bruker Daltonics, Germany) in the positive mode (target voltage 25 kV, pulsed ion extraction voltage 20 kV). The reflector voltage was set to 21 kV and the detector voltage to 17 kV. Peptide mass fingerprints (PMF) were calibrated against a standard mixture by assigning appropriate mono-isotopic masses to the peaks; that is, bradykinin (1–7), m/z 757.399; angiotensin I, m/z 1296.685; angiotensin II, m/z 1046.54; rennin-substrate, m/z 1758.93; ACTH clip (1–17), m/z 2093.086; and somatostatin, m/z 3147.471 (peptide calibration standard II, Bruker Daltonics, Germany). MS spectra were recorded automatically across the mass range m/z 700–3000 and spectra were typically the sum of 400 laser shots. The PMFs were processed using Flex AnalysisTM software (version 2.4, Bruker Daltonics, Germany) and the sophisticated numerical annotation procedure (SNAP) algorithms were used for peak detection (S/N, 3; maximum number of peaks, 100; quality factor threshold, 30). MS data were interpreted using BioTools v3.2 (Bruker Daltonics, Germany), together with the Mascot search algorithm (version 2.0.04 updated 09/05/2018; Matrix Science Ltd., UK). Mascot parameters were as follows: fixed cysteine modification with propionamide, variable modification due to methionine oxidation, one missed cleavage site (i.e., in the case of incomplete trypsin hydrolysis), and amass tolerance of 100 ppm. Identified proteins were accepted as correct if they showed a Mascot score greater than 56 and p < 0.05, sequence coverage of at least 20%, and a minimum of four matched peptides. Not all spots of interest could be identified because some proteins were of low abundance and did not yield sufficiently intense mass fingerprints, whereas others were mixtures of multiple proteins [48 (link)].
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Angiotensin I
Angiotensin II
Angiotensinogen
Bradykinin
Clip
Coumaric Acids
Cysteine
Cytokinesis
Exanthema
Fingerprints, Peptide
Hydrolysis
Immune Tolerance
Isotopes
Methionine
Peptides
propionamide
Proteins
Somatostatin
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Trypsin
Z1046
Most recents protocols related to «Propionamide»
Example 41
N-(3-(7-methyl-2-((4-(4-methylpiperazin-1-yl)phenyl)amino)quinazolin-8-yl)phenyl)propionamide (40.8 mg) was prepared as described for N-(3-(7-methyl-2-((4-morpholinophenyl)amino)quinazolin-8-yl)phenyl)acrylamide. LRMS (M+H+) m/z calculated 481.3, found 481.0. 1H NMR ((DMSO-d6, 300 MHz) δ 10.00 (s, 1H), 9.60 (s, 1H), 9.19 (s, 1H), 7.79-7.86 (m, 2H), 7.31-7.57 (m, 5H), 6.96 (d, 1H), 6.66 (d, 2H), 3.15-3.28 (m, 6H), 2.73-2.89 (m, 4H), 2.28-2.33 (m, 6H), 1.05 (t, 3H).
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1H NMR
Acrylamide
propionamide
Sulfoxide, Dimethyl
Changes in S-nitrosylation levels were analyzed with the Biotin switch assay as described by Jaffrey and Snyder [88 (link)]. Briefly, samples of 200 μg protein were treated with S-methyl methanethiosulfonate (MMTS, Sigma Aldrich, USA, Cat. #64,306) for 1 h at 50 °C in the dark to block cysteine-free thiols, and then, proteins were precipitated with ice-cold acetone for 24 h at − 20 °C. The S-nitrosylated cysteine residues were reduced with 2.5 mM sodium ascorbate (Sigma Aldrich, USA, Cat. #A7506) and 4 mM N-[6-(Biotinamido) hexyl]-3′-(2′-pyridyldithio) propionamide (HPDP-biotin, Thermo Scientific, Cat. #21,341) was used to label the reduced thiols with biotin. Samples were incubated for 1 h with agarose-conjugated streptavidin beads (Thermo Scientific, Cat. #20,353) and centrifuged to pull down HPDP-biotinylated proteins, which were finally separated by SDS-PAGE to be detected with specific antibodies.
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N-(2-Hydroxyethyl)acrylamide (HEAA, 99%); N,N’-Methylenebisacrylamide (MEAA, 99%); 1-Vinylimidazole (VI, 99%); 1-Vinylimidazolium bromide (VIBr, 99%); 2,2′-Azobis(2-methylpropionitrile) (AIBN, 99%); 2,2′-Azobis(2-methyl-N-(2-hydroxyethyl)propionamide) (VA-086, 99%); Dopamine hydrochloride (99%); Gelatin (CP); Bromoethane (99%); 3-Mercaptopropionic acid (99%); Triton-X 100 (99%); and Polyoxymethylene (99%) were procured from Aladdin. Four-armed polyethylene glycol (4-PEG, 5K) was obtained from SINOPEG Co. E. coli (DH5α) and S. aureus (ATCC 25923) were sourced from Beyotime. Human Skin Fibroblast (HSF), Human Lymphoma Cell (U-937), Human Umbilical Vein Endothelial Cells (HUVEC), PRMI-160 Culture Medium, Special DMEM high glucose medium, DMEM Culture Medium, L13152 LIVE/DEAD® Bac LightTM Bacterial Viability Kit, Cell Counting Kit-8 (CCK8), Matrigel Matrix Gel, Lipopolysaccharide (LPS) were purchased from Thermo Fisher Scientific. Rhodamine B and 4,6-Diamidino-2-phenylindole (DAPI) were procured from Cell Signaling Technology.
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Protein solutions were prepared by ice-cold NP-40 buffer containing 1% protease inhibitors and collected by centrifuging under 14000 rpm for 20 min. N-ethylmaleimide (NEM, 20 mM) was added to protein solutions of equal concentration, and the samples were incubated at 4 °C for 2 h. Methanol precipitation was performed three times and the pellet was resuspended in solubilisation buffer (4% SDS, 50 mM Tris·HCl, 5 mM EDTA, pH 7.4) for incubation at room temperature for 5 min. Hydroxylamine (HA, pH 7.4, 25% final concentration) and EZ-link® N-[6-(biotinamido) hexyl]-3′ -(2′ -pyridyldithio)propionamide (biotin-HPDP) were added to incubate with protein solutions at 4 °C overnight. Avidin agaroses were washed by PBS twice and interacted with the mixture. SDS loading buffer was used to collect the remained proteins and western blotting was performed to analyze the captured PKCδ.
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To understand the binding mechanism between designed peptides and thrombin, molecular dynamics simulation was carried out. In this study, the designed peptide binder in docking complex with human thrombin, and human thrombin carried its native binder (2-[2-(4-chloro-phenylsulfanyl)-acetylamino]-3-(4-guanidino-phenyl)-propionamide) were used for MD simulation. The docking complex was independently subjected to MD simulation using Gromacs-2020,[28 ] and the simulation system was filled with SPC/E water in a cubic box. The system was neutralized using Na+ and Cl−, and the distance between the edge of water box and docking complex was 15 Å. The system undergoes energy minimization using the steepest descent method, followed by equilibration using the isochoric–isothermal ensemble and isothermal–isovolumetric ensemble under 300 K for 100 and 200 ps, respectively. The production of MD simulation was carried out for 100 ns under 300 K, and we used the last 10 ns of simulation for gmxMMPBSA[29 (link)] analysis.
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Proteome Discoverer is a software solution for the analysis of mass spectrometry-based proteomic data. It provides a comprehensive platform for the identification, quantification, and characterization of proteins from complex biological samples.
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N-(6-(Biotinamido) hexyl)-3'-(2′-pyridyldithio)-propionamide (HPDP)-biotin is a heterobifunctional crosslinking reagent that contains a biotin group and a pyridyldithio reactive group. It is commonly used in protein labeling and modification applications.
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The Mascot Server is a software application that provides protein identification and characterization services. It is designed to analyze mass spectrometry data and match it against protein sequence databases to identify the proteins present in a sample. The Mascot Server operates as a centralized system, allowing multiple users to access and utilize the software's capabilities simultaneously.
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Acetonitrile is a colorless, volatile, flammable liquid. It is a commonly used solvent in various analytical and chemical applications, including liquid chromatography, gas chromatography, and other laboratory procedures. Acetonitrile is known for its high polarity and ability to dissolve a wide range of organic compounds.
The AB SCIEX MS Data Converter beta 1.1 is a software tool designed to convert mass spectrometry data files from various formats into a universal format. This software is currently in the beta testing phase.
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Trypsin is a serine protease enzyme that is commonly used in cell culture and molecular biology applications. It functions by cleaving peptide bonds at the carboxyl side of arginine and lysine residues, which facilitates the dissociation of adherent cells from cell culture surfaces and the digestion of proteins.
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Propionamide is a chemical compound used in various laboratory applications. It serves as a reagent and intermediate in organic synthesis. Propionamide is a colorless, crystalline solid with a melting point of approximately 79°C. Its chemical formula is CH3CH2CONH2.
More about "Propionamide"
Propionamide is a synthetic organic compound with the chemical formula CH3CH2CONH2.
It is a versatile molecule with a range of applications in research and industry.
Propionamide is commonly used as an intermediate in the production of other chemicals, making it an important compound for various industrial processes.
Propionamide research is crucial for understanding its properties, potential uses, and optimizing production methods.
Researchers studying Propionamide can leverage AI-driven platforms like PubCompare.ai to enhance the reproducibility and accuracy of their work.
PubCompare.ai helps researchers locate relevant protocols from literature, preprints, and patents, and provides AI-driven comparisons to identify the best protocols and products.
This can be particularly useful when working with related compounds and techniques, such as Proteome Discoverer, N-(6-(Biotinamido) hexyl)-3'-(2′-pyridyldithio)-propionamide (HPDP)-biotin, Mascot server, Acetonitrile, AB SCIEX MS Data Converter beta 1.1, Mascot, Trolox, Formic acid, and Trypsin.
By optimizing Propionamide research and development efforts, researchers can unlock new applications and improve the efficiency of related processes.
Propionamide's versatility and importance in various fields make it a key compound for further exploration and innovation.
With the help of AI-driven platforms like PubCompare.ai, researchers can enhance the reproducibility and accuracy of their Propionamide studies, ultimately driving progress in this dynamic area of research and development.
It is a versatile molecule with a range of applications in research and industry.
Propionamide is commonly used as an intermediate in the production of other chemicals, making it an important compound for various industrial processes.
Propionamide research is crucial for understanding its properties, potential uses, and optimizing production methods.
Researchers studying Propionamide can leverage AI-driven platforms like PubCompare.ai to enhance the reproducibility and accuracy of their work.
PubCompare.ai helps researchers locate relevant protocols from literature, preprints, and patents, and provides AI-driven comparisons to identify the best protocols and products.
This can be particularly useful when working with related compounds and techniques, such as Proteome Discoverer, N-(6-(Biotinamido) hexyl)-3'-(2′-pyridyldithio)-propionamide (HPDP)-biotin, Mascot server, Acetonitrile, AB SCIEX MS Data Converter beta 1.1, Mascot, Trolox, Formic acid, and Trypsin.
By optimizing Propionamide research and development efforts, researchers can unlock new applications and improve the efficiency of related processes.
Propionamide's versatility and importance in various fields make it a key compound for further exploration and innovation.
With the help of AI-driven platforms like PubCompare.ai, researchers can enhance the reproducibility and accuracy of their Propionamide studies, ultimately driving progress in this dynamic area of research and development.