Acquity uplc beh300 c4 column

Manufactured by Waters Corporation

Sourced in Germany

The Acquity UPLC BEH300 C4 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of large biomolecules, such as proteins and peptides. The column features a bonded C4 stationary phase, which provides excellent resolution and peak shape for these types of analytes. The column is compatible with ultra-performance liquid chromatography (UPLC) systems and offers fast, efficient separations with high sensitivity.

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Lab products found in correlation

Acquity uplc esi tqd, by Waters Corporation (4 mentions) Maxent 1, by Waters Corporation (1 mentions) Acquity uplc 1 class system, by Waters Corporation (1 mentions) Waters select series cyclic ims, by Waters Corporation (1 mentions) Masshunter software, by Agilent Technologies (1 mentions) Agilent 1290, by Agilent Technologies (1 mentions) Tissuelyser, by Qiagen (1 mentions) Qtrap 5500 mass spectrometer, by AB Sciex (1 mentions) Multiquant software, by AB Sciex (1 mentions) Acquity uplc system, by Waters Corporation (1 mentions)

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Acquity UPLC (716 citations) C4 column (9 citations) BEH300 C4 column (7 citations) Acquity BEH300 C4 column (4 citations)

9 protocols using acquity uplc beh300 c4 column

Reverse-phase LC-MS Analysis of Intact Proteins

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Reverse-phase LC–MS analysis of the intact proteins were performed on a Waters Acquity BEH300 C4 UPLC column (2.1 × 150 mm, 1.7 μm) under the following parameters: mobile phase “A”: 0.1% trifluoroacetic acid in water, mobile phase “B”: 0.1% trifluoroacetic acid in acetonitrile; flow rate: 400 μl/min; column temperature: 80 °C; gradient: 1 min: 5% B, 12 min: 50% B, and 12.5 min: 90% B. UV detection was performed at 220 and 280 nm. The m/z range was 400 to 2000. Deconvolution was performed by the MaxEnt 1 software (Waters Corporation).

Vadászi H., Kiss B., Micsonai A., Schlosser G., Szaniszló T., Kovács R.Á., Györffy B.A., Kékesi K.A., Goto Y., Uzonyi B., Liliom K, & Kardos J. (2022). Competitive inhibition of the classical complement pathway using exogenous single-chain C1q recognition proteins. The Journal of Biological Chemistry, 298(7), 102113.

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Reverse-Phase LC-MS Analysis of Intact Proteins

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Reverse-phase LC-MS analyses of intact proteins were performed on a Waters Acquity I-Class UPLC system coupled directly to a high-resolution hybrid quadrupole-time-of-flight mass spectrometer (Waters Select Series Cyclic IMS; Waters Corporation (Milford, MA, USA)). Samples were analyzed using a Waters Acquity BEH 300 C4 UPLC column (2.1 × 150 mm, 1.7 μm) under the following parameters: mobile phase “A”: 0.1% trifluoroacetic acid in water; mobile phase “B”: 0.1% trifluoroacetic acid in acetonitrile; flow rate: 300 μL/min; column temperature: 60 °C; gradient: 2 min: 5% B, 8 min: 55% B, and 8.5 min: 90% B. UV detection was performed at 220 nm and 280 nm. The spectrometer was operated in ESI positive V mode. Leucine encephalin was used as a lock mass standard. The m/z range was 900–2000. Deconvolution was performed by MaxEnt 1 software (MassLynx v4.2, Waters Corporation).

Nagy-Fazekas D., Stráner P., Ecsédi P., Taricska N., Borbély A., Nyitray L, & Perczel A. (2023). A Novel Fusion Protein System for the Production of Nanobodies and the SARS-CoV-2 Spike RBD in a Bacterial System. Bioengineering, 10(3), 389.

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Ras GTPase Modification Kinetics

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200 μL reactions were made in Gel Filtration buffer (20 mM HEPES (pH 7.5), 150 mM NaCl) with no added reductant at room temperature. The final concentrations of each reagent were as follows: 4 μM Ras, 1 mM MgCl₂, 20 μM Compound 3, X μM Compound 4, and a total DMSO concentration of 4% by volume. Compound 4 was varied from 0X (0 μM), 1X (20 μM), 3X (60 μM), and 9X (180 μM) respectively. Adding the desired volume of Gel Filtration Buffer, MgCl₂, DMSO, and 10 mM compound stocks of Compounds 3 and 4 was done first and mixed thoroughly before protein was added. Reactions were started by the addition and gentle mixing by pipette of concentrated Ras stocks diluted into the master reaction down to 4 μM. Each reaction was set in triplicate and allowed to react at room temperature in the dark (either covered in aluminum foil or in a bench drawer) until the desired time point was reached. At each time point, 22.5 μL aliquots of each 200 μL master reaction was sampled and quenched by the addition of 2.5 μL of 2% formic acid (also made in Gel Filtration Buffer). This 25 μL quenched sample was then analyzed by LC/MS and the remaining master reaction was allowed to continue reacting after gentle mixing by pipette. The percent modification was analyzed by electrospray mass spectrometry using a Waters Acquity UPLC/ESI-TQD with a 2.1 × 50 mm Acquity UPLC BEH300 C4 column.

Gentile D.R., Rathinaswamy M.K., Jenkins M.L., Moss S.M., Siempelkamp B.D., Renslo A.R., Burke J.E, & Shokat K.M. (2017). Ras Binder Induces a Modified Switch-II Pocket in GTP- and GDP-States. Cell chemical biology, 24(12), 1455-1466.e14.

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Nano-LC-ESI-QTOF Mass Spectrometry

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Samples were separated on an Acquity UPLC BEH300 C4 column (2.1 × 100 mm, 300 Å, 1.7 μm; Waters) attached to an Agilent 1290 chromatographic system (Agilent Technologies, Waldbronn, Germany). The column temperature was set to 30°C, the flow rate was 0.2 ml/min and the gradient of solvent A (0.01% TFA in H₂O) and solvent B (0.01% TFA in ACN) was 5% B from 0–3 min, 5–95% B during 3–18 min, hold 95% B from 18–21 min, re-equilibrate column with 5% B from 21.1 to 28 min. From 6 to 28 min, the flowpath was switched to an online coupled ESI–QTOF 6520 (Agilent Technologies) mass spectrometer. MS data were recorded in positive mode and evaluated using the MassHunter software (Agilent Technologies).

Pech A., Achenbach J., Jahnz M., Schülzchen S., Jarosch F., Bordusa F, & Klussmann S. (2017). A thermostable d-polymerase for mirror-image PCR. Nucleic Acids Research, 45(7), 3997-4005.

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Lipidomic Analysis of Mouse Adipose Tissue

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All mouse adipose tissue samples were homogenized in methanol:water (1:1, v/v) using the Qiagen tissue lyser. Following homogenization, lipids were extracted from 5 μL homogenate with 2000 μL of the extraction solvent: dichloromethane:isopropanol:methanol (25:10:65, v/v/v). Lipid extracts were then analyzed by UPLC-MS/MS using a Waters Acquity UPLC coupled to a Sciex QTRAP 5500 mass spectrometer. Lipid classes were separated by reversed-phase chromatography on a Waters Acquity UPLC BEH300 C4 column, 1.7 μm, 2.1 × 50 mm. Lipid species were then analyzed on the mass spectrometer using positive ion electrospray ionization in the multiple reaction-monitoring (MRM) mode. LC chromatogram peak integration was performed with Sciex MultiQuant software. All data reduction was performed with in-house software in Pfizer Inc.

Li M.D., Vera N.B., Yang Y., Zhang B., Ni W., Ziso-Qejvanaj E., Ding S., Zhang K., Yin R., Wang S., Zhou X., Fang E.X., Xu T., Erion D.M, & Yang X. (2018). Adipocyte OGT governs diet-induced hyperphagia and obesity. Nature Communications, 9, 5103.

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Proteolysis and Mass Spectrometry Analysis

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In selected conditions, proteolysis reactions were performed as described above in a volume of 500 μL. After quenching with EDTA, samples were concentrated to 100 μL and submitted to two concentration/dilution cycles with water to a final volume of 100 μL using Vivaspin 10000 Da cut-off filters. These samples were analyzed by high performance liquid chromatography/electrospray-ionization mass spectrometry (HPLC/ESI-MS) at the high resolution mass spectrometry service at the Centro de Instrumentación Científica, University of Granada. HPLC/ESI-MS was performed in a Acquity UPLC system (Waters) using a gradient of water/formic acid (0.1%) and acetonitrile/formic acid (0.1%) using a Acquity UPLC BEH300 C4 column (2.1 × 50 mm; Waters) coupled to a QTOF Synapt62 HDMS (Waters).
For N-terminal sequencing, 100 μL of selected proteolysis mixtures were denatured in Laemmli´s buffer and run in a 12% SDS-PAGE followed by electrotransference to a polyvinylidene difluoride (PVDF) membrane. The membrane was stained with Coomassie blue G-250 and selected bands were cut, destained and equilibrated in water for N-terminal sequencing by the Edman´s method (performed at the service of Protein Chemistry, Centro de Investigaciones Biológicas, Madrid, Spain).

Medina-Carmona E., Palomino-Morales R.J., Fuchs J.E., Padín-Gonzalez E., Mesa-Torres N., Salido E., Timson D.J, & Pey A.L. (2016). Conformational dynamics is key to understanding loss-of-function of NQO1 cancer-associated polymorphisms and its correction by pharmacological ligands. Scientific Reports, 6, 20331.

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K-Ras Compound Binding Assay

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50uL of 4uM K-Ras^G12C1–169 or K-Ras^WT 1–189 was incubated with 200uM or 25uM compound (4 or 2% v/v dimethylsulfoxide respectively) for 24 hours at room temperature. The reaction was quenched with 2uL 10% v/v formic acid to yield 0.4% v/v formic acid final. Mass spectrometry experiments were performed using the Waters Acquity UPLC/ESI-TQD with a 2.1 × 50 mm Acquity UPLC BEH300 C4 column.

Nnadi C.I., Jenkins M.L., Gentile D.R., Bateman L.A., Zaidman D., Balius T.E., Nomura D.K., Burke J.E., Shokat K.M, & London N. (2018). Novel K-Ras G12C Switch-II Covalent Binders Destabilize Ras and Accelerate Nucleotide Exchange. Journal of chemical information and modeling, 58(2), 464-471.

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Ras Protein Modification by Beta-Mercaptoethanol

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Each reaction was 50 μL total volume and conducted in a 96 well plate format for analysis by LC/MS. A base master mix of 4 μM Ras, 1 mM MgCl₂, 100 μM tethering compound in Gel Filtration Buffer was made. Another master mix containing the same reagents supplemented with 25 mM βME was also made. 100 μL of the master mix containing 25 mM βME was put in wells in row A. The remaining rows were filled with 50 μL of the master mix with no βME. Using a multi-channel pipette, the solutions were serial diluted 1 : 1 from row A down to H. This made 8 reactions with βME concentration varying from 25 mM down to 185 μM. Once set, the tray was allowed to equilibrate while mixing at room temperature for 1 h. After equilibration, the percent modification was detected by LC/MS. The percent modification was analyzed by electrospray mass spectrometry using a Waters Acquity UPLC/ESI-TQD with a 2.1 × 50 mm Acquity UPLC BEH300 C4 column. Percent modifications for each βME concentration were plotted in PRISM and fit using a Boltzman sigmoidal non-linear regression (curve fit) to determine the βME₅₀ value and 95% confidence interval.

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High-throughput Disulfide-trapping Screening

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The disulfide-trapping screen was performed according to standard procedures. Purified LRH-1 (Cys311Ser, Cys487Ser) was diluted to 4 μM in a buffer containing 25 mM HEPES, 150 mM NaCl and 500 μM BME. The protein was aliquoted into a 96 well-plate (25 μL per well) and individually incubated at ambient temperature for 1 h with disulfide-linked monophores (1280 total compounds) at 150 μM. After the equilibration period, reaction mixtures were analyzed by high-throughput mass spectrometry using Waters Acquity UPLC/ESI-TQD with a 2.1 x 50mm Acquity UPLC BEH300 C4 column. The extent of labeling was determined by comparing the molecular masses of the covalently adducted protein to the molecular mass of the free protein. Hits were defined as those compounds that conjugated with efficiencies greater than two standard deviations (SD) above the mean.

de Jesus Cortez F., Suzawa M., Irvy S., Bruning J.M., Sablin E., Jacobson M.P., Fletterick R.J., Ingraham H.A, & England P.M. (2016). Disulfide-Trapping Identifies a New, Effective Chemical Probe for Activating the Nuclear Receptor Human LRH-1 (NR5A2). PLoS ONE, 11(7), e0159316.

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