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Itraq kit

Manufactured by Thermo Fisher Scientific
Sourced in United States

The iTRAQ kit is a multiplexed quantitation kit designed for comparative proteomic analysis. The kit utilizes isobaric tags that are covalently attached to the N-terminus and lysine residues of peptides from different samples. The tagged peptides are then analyzed by mass spectrometry, allowing for the relative quantification of proteins across multiple samples simultaneously.

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9 protocols using itraq kit

1

Quantitative Proteome Analysis via iTRAQ

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According to the instructions of the iTRAQ kit (Applied Biosystems, Carlsbad, CA, United States), 100 μg of the reduction reagent was added to each sample followed by incubation at 60°C for 1 h. Next, cysteine was added as a blocking reagent. After incubation for 10 min at room temperature, precooled acetone (acetone: sample = 5:1, v/v) was added to each tube followed by precipitation at −20°C for 1 h. Next, the samples were centrifuged at 12,000 rpm for 20 min at 4°C, and 20 μL of the dissolution solution was added to the precipitate to resuspend and dissolve the sample. Trypsin (Sigma, CA, United States) was added at a ratio of 1:20 (enzyme: protein), and this mixture was incubated overnight at 37°C. Labeling reagent was added to each tube and mixed. After reaction of the samples at room temperature for 1 h, 3 volumes of water were added.
The peptide mixture was then fractionated on a surveyor HPLC system with a 1.7 μm Waters BEH C18 2.1 × 50 mm reversed-phase column (Waters Company, CA, United States). The chromatographic method was as follows: the peptides were eluted with a 5–35% B (A: 20 mM ammonium formate, pH = 10.0; B: acetonitrile) gradient for 16 min. Absorbance was measured at 214 nm, and a total of 40 fractions were collected and then combined into 10 fractions.
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2

Peptide Labeling with iTRAQ

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Using the iTRAQ kit (Applied Biosystems, Foster City, CA) protocol, the peptides were labeled with iTRAQ tags as follows: NAT, iTRAQ 118 (IT118); CA, iTRAQ 121 (IT121); LN, iTRAQ 119(IT119). The labeled samples were mixed and dried by a rotary vacuum concentrator (Christ RVC 2-25; Osterode am Harz, Germany).
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3

Protein Digestion and Labeling Protocol

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Protein aliquots (100 μg) were digested into peptides using 3.3 μg Trypsin (Thermo Scientific.) at 37°C for 24 h, following with centrifugation (at 16000 g, 4°C, 10 min). Peptide precipitation was vacuum-dried, diluted in 0.5 M tetraethylammonium bromide, and then prepared using an iTRAQ kit (Applied Biosystems, Carlsbad, CA, United States) according to the recommendation from the manufacturer. The samples were pooled and vacuum-dried.
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4

Quantitative Proteomics by 8-plex iTRAQ

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Quantitative 8-plex iTRAQ proteomic analysis was carried out on eight independent protein samples. Total proteins were extracted with iTRAQ lysis buffer, processed by disulfide bond cleavage and reductive alkylation of proteins with DTT and iodoacetamide, and then the proteins were hydrolyzed by trypsin. The pooled samples were labeled using the iTRAQ kit according to the manufacturer's protocol (Applied Biosystems Sciex).
The iTRAQ-labeled samples were mixed and resuspended in buffer A (2% acetonitrile, pH 10) and eluted with a 2–95% linear gradient of buffer B (90% acetonitrile, pH 10) over 90 min with a flow rate of 1 mL/min.
The samples were separated into 20 fractions on a C18 column with the HPLC system (Thermo Fisher Scientific) and analyzed using a Q Exactive Plus hybrid quadrupole-orbitrap Mass Spectrometer (Thermo Fisher Scientific). The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD023048.
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5

Quantitative Proteomic Analysis of T2DM

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One hundred micrograms of proteins from each sample was reduced, alkylated, and digested with trypsin (Promega). The digested peptides were then dried and reconstituted in 50 μL 0.5 M triethyl ammonium bicarbonate (TEAB). Digested peptide samples were labeled using the iTRAQ kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. The iTRAQ tags were as follows: healthy control 1-iTRAQ 114; healthy control 2-iTRAQ 115; T2DM 1-iTRAQ 116; and T2DM 2-iTRAQ 117. The labeled samples were finally combined into one sample mixture and dried with a rotary vacuum concentrator.
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6

Proteomic Analysis Workflow

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The system in the MARS.14 chromatographic column was obtained from Aglient Co.; the iTRAQ kit, QSTAR XL mass spectrometer, and Protein Pilot4.2 software were obtained from Applied Biosystems (USA); the liquid chromatograph (20AD) was obtained from Shimadzu Co. (Japan); Strong Cation Exchange Chromatography (2.1×100 mm, 5 um, 300 A) was obtained for the Nest Group Inc. System (USA); and the ZORBAX 300SB-C18 column (5 um, 300A, 0.1×150 mm) was obtained from Microm Co. (USA).
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7

Quantitative Proteomics of Infection Samples

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Total protein samples were reduced and cysteine-blocked according to the protocol provided by the manufacturer (iTRAQ kit, Applied BioSystems). Samples were then 1:10 diluted in 0.5 M triethylammonium bicarbonate (pH 8.5). Each sample was treated with 1 μg trypsin (25 μl) and kept at 37°C overnight. After the samples were vacuum-dried, they were resuspended in 35 μl of 0.5 M triethylammonium bicarbonate (pH 8.5). The non-infected and infected samples were labeled with iTRAQ reagents 114 and 115, respectively. These labeled protein samples were pooled and then purified using a strong cation exchange column. The bound peptides were eluted with 5% NH4OH in 30% methanol.
After vacuum-dried, the iTRAQ-labeled peptides were reconstituted with 20 μl of 5 mM KH2PO4 containing 5% acetonitrile (pH 3.0), and separated via 2D-Liquid Chromatography (2D-LC) with an Ultimate Dual-gradient LC system (Dionex Ultimate3000).
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8

HSYA Isolation and Analysis in C. tinctorius

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HSYA was isolated and purified from the aqueous extract of C. tinctorius L. by macroporous resin-gel column chromatography, as described previously14 (link). The molecular weight of HSYA is 612. HSYA was analysed using a high-performance liquid chromatography system (Shimadzu, Kyoto, Japan). iTRAQ kits were purchased from Applied Biosystems (Waltham, MA, USA), the 2-D Quant Kit from GE Healthcare (Pittsburgh, PA, USA), sequence-grade modified trypsin from Promega (Madison, WI, USA), and human ox-LDL (BT-910) from Alfa Aesar (Heysham, Lancashire, UK). Aspirin was the product of Sigma-Aldrich (St. Louis, MO, USA). Bromophenol blue, tetramethylethylenediamine, Coomassie brilliant blue G-250, Bis, low-molecular weight marker, nitrocellulose membrane, Tris-base, and the Bradford method protein assay kit were purchased from Bio-Rad (Hercules, CA, USA). VDAC-2 polyclonal antibody was purchased from Cell Signaling Technology (Danvers, MA, USA), and GAPDH mouse monoclonal antibody was from Beyotime Biotechnology (Jiangsu, China). Other polyclonal and monoclonal antibodies and siRNA reagents were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), with the secondary antibodies obtained from LI-COR Biosciences (Lincoln, NE, USA). NO and MDA assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
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9

Peptide Labeling and Fractionation for Mass Spec

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The peptide samples were dissolved in 0.5 M TEAB and labelled using iTRAQ kits (Applied Biosystems, Foster City, CA). The mixed peptides were fractionated using the Ultimate 3000 HPLC system (Thermo DINOEX, United States). Mass spectrometry data were produced by the TripleTOF 5600 + liquid mass spectrometry system (SCIEX, United States) coupled with the Eksigent nanoLC system (SCIEX, United States). TripleTOF 5600plus liquid chromatography and mass spectrometry system (SCIEX) was used for mass spectrometry data acquisition.
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