Eksigent nanolc

Manufactured by AB Sciex

Sourced in United States

The Eksigent nanoLC is a high-performance liquid chromatography (HPLC) system designed for the separation and analysis of small-scale samples, such as those encountered in proteomics and lipidomics research. The core function of the Eksigent nanoLC is to provide precise and reproducible separation of complex mixtures at nanoliter flow rates, enabling the analysis of minute sample quantities.

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Lab products found in correlation

Trypsin, by Promega (2 mentions) Picotip emitter, by New Objective (1 mentions) Reprosil pur c18 resin, by Dr. Maisch (1 mentions) Glu c, by Promega (1 mentions) Sciex 5600, by AB Sciex (1 mentions) Trypsin gold, by Promega (1 mentions) Orbitrap elite mass spectrometer, by Thermo Fisher Scientific (1 mentions) Laemmli buffer, by Bio-Rad (1 mentions) Imperial protein stain, by Thermo Fisher Scientific (1 mentions) Mascot software, by Matrix Science (1 mentions) Ltq orbitrap velos pro, by Thermo Fisher Scientific (1 mentions) Iodoacetamide, by Fujifilm (1 mentions) Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions) Trifluoroacetate, by Merck Group (1 mentions) C18 spin column, by Thermo Fisher Scientific (1 mentions) Proteinpilot software, by AB Sciex (1 mentions) Triple tof 5600 system, by AB Sciex (1 mentions) Q exactive, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Eksigent nanoLC-1D plus system Eksigent nanoLC system Eksigent nanoLC 400 liquid chromatography system Eksigent NanoLC 400 System Eksigent nanoLC 415 nanoscale RP-HPLC Eksigent NanoLC Ultra® system Eksigent nanoLC Ultra 2D plus system Eksigent nanoLC‐Ultra 1D+ Eksigent NanoLC-425 Eksigent nanoLC-Ultra 2D

6 protocols using eksigent nanolc

SILAC Proteomics of AMPK Activator

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Proteomic experiments with AMPK activator PF-06409577 were carried out with triple SILAC labeled HepG2 cells (Ong and Mann, 2007 ). HepG2 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in customized high glucose SILAC DMEM media (PAA laboratories, catalog number E15-086) supplemented with 10% dialyzed FBS with 10 K molecular weight cut off (PAA laboratories, catalog number A11-507) and supplemented with penicillin/streptomycin. Extracted proteins were quantified by Bradford method and combined at equal protein concentration (1,1:1 light:medium:heavy). Proteins were separated using 1D-SDS gel and subdivided into 12 gel slices. For each gel slice, proteins were first reduced with dithiothreitol (DTT) and alkylated with iodoacetamide (IAA) before proceeding with trypsin digestion which was carried out at 37 °C for overnight.
Mass spectrometric analyses were carried out with Eksigent nanoLC (Sciex, Framingham, MA) coupled with Elite LTQ-Orbitrap mass spectrometer (Thermo, Bremen, German). Nano-LC column (15 cm × 75 μm) was packed in house using precut silica tubing PicoTip Emitter (New Objective, Woburn, MA) with 3 μm ReproSil-Pur C18 resin (Dr. Maisch, Entringen, Germany).

Esquejo R.M., Salatto C.T., Delmore J., Albuquerque B., Reyes A., Shi Y., Moccia R., Cokorinos E., Peloquin M., Monetti M., Barricklow J., Bollinger E., Smith B.K., Day E.A., Nguyen C., Geoghegan K.F., Kreeger J.M., Opsahl A., Ward J., Kalgutkar A.S., Tess D., Butler L., Shirai N., Osborne T.F., Steinberg G.R., Birnbaum M.J., Cameron K.O, & Miller R.A. (2018). Activation of Liver AMPK with PF-06409577 Corrects NAFLD and Lowers Cholesterol in Rodent and Primate Preclinical Models. EBioMedicine, 31, 122-132.

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Proteomic Analysis of Nostoc Cyanobacteria

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Freeze-dried, ice-affinity purified material was sent to the Medical University of South Carolina (MUSC) and later to the College of Charleston (Charleston, SC), for liquid chromatography/mass spectrometry (LC/MS) analysis of tryptic peptides. Approximately 20 μg of total protein (as estimated from gel electrophoresis) was digested overnight at 37°C with trypsin gold (1:20; Promega) or GluC (1:10; Promega). Peptides were separated using two LC/MS platforms, an Eksigent nano-LC coupled to a SCIEX 5600 with a nanospray source and a Dionex Ultimate 3000 nano-LC coupled to an Orbitrap Fusion Lumos with a nanospray source at Hollings Marine Laboratory (Charleston, SC). Data were acquired from the Eksigent platform as described previously (39 (link)) and from the Dionex platform as described previously (40 (link)). Masses of the tryptic peptides were compared with predicted tryptic peptides in the Nostoc contig file translated in all six frames using MASCOT and MS-GF+ search engines. Searches included standard variable modifications and carbamidomethyl-fixed modifications. The search parameter Enzyme was set to trypsin or semitrypsin for data from trypsin digests or GluC(DE) for GluC endopeptidase digests. Error-tolerant searches were conducted to look for modified peptides not otherwise included.

Raymond J.A., Janech M.G, & Mangiagalli M. (2021). Ice-Binding Proteins Associated with an Antarctic Cyanobacterium, Nostoc sp. HG1. Applied and Environmental Microbiology, 87(2), e02499-20.

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Mass Spectrometry Analysis of Phage Proteins

Cited 1 time

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The purified and concentrated phage lysate was subjected to gel electrophoresis, in-gel digestion with Trypsin, reverse phase nanoflow high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS-MS), and data analyses as previously described (42 (link)). In brief, the CsCl-purified phage lysate was reduced with 0.5% 2-mercaptoethanol in Laemmli buffer per manufactures' instructions (Bio-Rad, Hercules, CA, USA) and subsequently subjected to SDS-PAGE using a 1D Bio-Rad 12% TGX gel (Bio-Rad, Hercules, CA, USA). The gel was stained using Imperial^TM Protein Stain (ThermoFisher, Waltham, MA, USA). Protein bands were excised and subsequently digested in gel with Trypsin (Promega, Madison, WI, USA) using a Digest Pro robot (Intavis, Köln, Germany). Sample digests were subjected to nanoflow reversed-phase chromatography with an Eksigent NanoLC (Sciex, Framingham, MA, USA) using Picochip columns (New Objectives, Woburn, MA, USA). Tandem mass spectra (MS-MS) were obtained in positive ion mode with an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Mascot software (Matrix Science, Boston, MA, USA) was used to match the MS-MS data to amino acid sequences derived from nucleotide sequences previously obtained from the phage isolates.

Liao Y.T., Zhang Y., Salvador A., Harden L.A, & Wu V.C. (2022). Characterization of a T4-like Bacteriophage vB_EcoM-Sa45lw as a Potential Biocontrol Agent for Shiga Toxin-Producing Escherichia coli O45 Contaminated on Mung Bean Seeds. Microbiology Spectrum, 10(1), e02220-21.

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Quantitative Proteomic Analysis of CK2 Substrates

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After tryptic digestion, resulting peptides from treated and untreated samples were isotopically labelled with N-acetoxy-D₃-succinimide (D₃-NAS) and N-acetoxy-H₃-succinimide (H₃-NAS), respectively, and then pooled together. Finally, proteins were identified by LC-MS/MS analysis using Eksigent nanoLC (AB SCIEX, Framingham, MA, USA) coupled to LTQ Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Protein–protein interaction networks were constructed using information from the STRING database [47 (link)], and protein kinase CK2 substrates were identified using post-translational modification resource iPTMnet, web-based tool KEA2, and literature search [48 (link),49 (link)].

Rosales M., Pérez G.V., Ramón A.C., Cruz Y., Rodríguez-Ulloa A., Besada V., Ramos Y., Vázquez-Blomquist D., Caballero E., Aguilar D., González L.J., Zettl K., Wiśniewski J.R., Yang K., Perera Y, & Perea S.E. (2021). Targeting of Protein Kinase CK2 in Acute Myeloid Leukemia Cells Using the Clinical-Grade Synthetic-Peptide CIGB-300. Biomedicines, 9(7), 766.

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Mass Spectrometry Proteome Analysis

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RNF183 precipitates obtained as described in the previous section were used as the proteome analysis sample. Precipitate-bound beads were suspended in 25.5 μl of bicarbonate ammonium (21.25 mM, Wako Pure Chemical Industries). After adding 1.5 μl of dithiothreitol (12.5 mM, Thermo Scientific), the mixture was incubated at 95°C for 5 min. After cooling to room temperature, 3 μl of iodoacetamide (25 mM, Wako Pure Chemical Industries) were added, and the mixture was incubated at room temperature for 20 min. Next, 10 μl of 30 ng/μl trypsin (Promega Corporation) were added, and the mixture was incubated at 37°C for 3 h. Another 10 μl of trypsin were added, and the mixture was incubated overnight at 30°C. Finally, 2.5 μl of trifluoroacetate (Sigma-Aldrich) were added to terminate the reaction, and the sample was desalted using a C18 Spin Column (Thermo Fisher Scientific), followed by concentration for 2 h on a SpeedVac. Concentrates were analyzed on a TripleTOF 5600+ System with Eksigent nanoLC (AB SCIEX, Framingham, MA, USA). Proteins were identified using the ProteinPilot Software (AB SCIEX).

Wu Y., Guo X.P., Kanemoto S., Maeoka Y., Saito A., Asada R., Matsuhisa K., Ohtake Y., Imaizumi K, & Kaneko M. (2018). Sec16A, a key protein in COPII vesicle formation, regulates the stability and localization of the novel ubiquitin ligase RNF183. PLoS ONE, 13(1), e0190407.

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Quantitative Proteomics by DIA-MS

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Samples were measured on a QExactive (Thermo Fisher Scientific, Waltham MA, USA). Peptides were separated with an Eksigent NanoLC (AB Sciex, Washington, USA). We used a single-pump trapping 75-um scale configuration (Waters). 1ul of each were injected. Trapping was performed on a nanoEase TM symmetry C18 column (pore size 100Å, particle size 5um, inner diameter 180um, length 20mm). For separation, a nanoEase TM HSS C18 T3 column was used (pore size 100Å, particle size 1.8um, inner diameter 75um, length 250mm, heated to 50°C). Peptides were separated using a 120 min long linear solvent gradient of 5-35% ACN / 0.1% FA (using a flowrate of 300nl / min). Electronspray ionization with 2.6kV was used and a DIA method with a MS1 in each cycle followed by 35 fixed 20 Da precursor isolation (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
windows within a precursor range of 400-1100 m/z was applied. For MS1 we used a maximum injection time of 200ms and an AGC target of 3e6 with a resolution of 60k in the range of 350-1500 m/z. MS2 spectra were acquired using a maximum injection time of 55ms an AGC target of 1e6 with a 30k resolution. A collision energy of 28 was used for fragmentation.

Colameo D., Rajman M., Soutschek M., Bicker S., Ziegler L.v., Bohacek J., Germain P., Dieterich C., & Schratt G. (2020). Pervasive compartment-specific regulation of gene expression during homeostatic synaptic scaling.

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