Itraq 8 plex kit

Manufactured by AB Sciex

Sourced in United States

The iTRAQ 8-plex kit is a set of reagents used for quantitative proteomic analysis. It enables the simultaneous identification and relative quantification of proteins in up to 8 different samples. The kit utilizes isobaric tags that are covalently attached to the N-terminus and side chain amines of peptides, allowing for precise comparison of protein abundance across multiple biological conditions.

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Lab products found in correlation

Trypsin gold, by Promega (3 mentions) Strata x c18 column, by Phenomenex (2 mentions) Q exactive mass spectrometer, by Thermo Fisher Scientific (2 mentions) Acetonitrile, by Thermo Fisher Scientific (1 mentions) Formic acid, by Thermo Fisher Scientific (1 mentions) Nano pure system, by Thermo Fisher Scientific (1 mentions) Ammonium bicarbonate nh4hco3, by Merck Group (1 mentions) Iodoacetamide, by Merck Group (1 mentions) Urea, by Merck Group (1 mentions) Dithiothreitol (dtt), by Merck Group (1 mentions) Bovine pancreas tpck treated trypsin, by Merck Group (1 mentions) Proteome discoverer software version 1, by Thermo Fisher Scientific (1 mentions) L 3000 system, by Rigol (1 mentions) Lc 20ab hplc system, by Shimadzu (1 mentions) Ultremex scx column, by Phenomenex (1 mentions) Sequencing grade trypsin, by Promega (1 mentions) Trypsin, by AB Sciex (1 mentions) Nanospray 3 source, by AB Sciex (1 mentions) Proteinpilot software, by AB Sciex (1 mentions) Triple tof 5600 system, by AB Sciex (1 mentions)

28 protocols using itraq 8 plex kit

Xenopus laevis Embryo Proteomics

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Bovine pancreas TPCK-treated trypsin, urea, ammonium bicarbonate (NH₄HCO₃), dithiothreitol (DTT), and iodoacetamide (IAA) were purchased from Sigma–Aldrich (St. Louis, MO). Acetonitrile (ACN) and formic acid (FA) were purchased from Fisher Scientific (Pittsburgh, PA). Water was deionized by a Nano Pure system from Thermo scientific (Marietta, OH). iTRAQ 8-plex kits were purchased from AB Sciex (Foster City, CA).
Xenopuslaevis was purchased from Nasco (Fort Atkinson, WI). Mammalian Cell-PE LB™ buffer for embryo lysis was purchased from G-Biosciences (St. Louis, MO). Complete, mini protease inhibitor cocktail (provided in EASYpacks) was purchased from Roche (Indianapolis, IN).

Sun L., Bertke M.M., Champion M.M., Zhu G., Huber P.W, & Dovichi N.J. (2014). Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development. Scientific Reports, 4, 4365.

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Quantitative Proteomics Analysis by iTRAQ

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First, iTRAQ tagging and analysis was performed. The protein from each sample was reduced, alkylated and digested with trypsin. The digested peptides were dried and reconstituted in 50 µl 1 M triethylammonium bicarbonate. The dried peptides were labeled following the manufacturer's protocols (iTRAQ 8-plex kits; AB Sciex LLC, Framingham, MA, USA). Second, all the labelled peptides were pooled together followed by the separation of fractions using reversed-phase liquid chromatography which was performed by a RIGOL L-3000 system (RIGOL Technologies, Inc., Beijing, China). Then the fractionated peptides were analyzed using a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) fitted with a nano-liquid chromatography system. Then Proteome Discoverer software version 1.3 (Thermo Fisher Scientific, Inc.) was used to interpret the data files. The files were searched using the Mascot search engine against the human protein database downloaded from NCBI (www.ncbi.nlm.nih.gov, Refseq. Human.20130704. fasta).

Ding S., Liu C., Li Y., Liu H., Liu Z., Chen T., Zhang T., Shao Z, & Fu R. (2018). Expression of C1q in the serum of patients with non-severe aplastic anemia, and its association with disease severity. Molecular Medicine Reports, 19(2), 1194-1202.

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Quantitative Proteomics of Plant Flowers

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Three independent biological replicates performed in our experiment. Three internal standards (IS-1, IS-2, and IS-3) were prepared by mixing one biological replicate from the six tested samples. Protein extraction performed using TCA (trichloroacetic acid)-acetone method with some modifications.^{41 (link)} Flowers were ground to powder in liquid nitrogen using a mortar and pestle. The powder was mixed with cold acetone which contains 10% (w/v) TCA and 0.07% (w/v) 2-mercaptoethanol and then precipitated for 2 h at −20 °C. After centrifuging at 25 000 × g for 30 min, the pellet was washed with cold acetone twice and suspended in buffer containing 8 M urea, 4% CHAPS, 2% ampholyte and 20 mM DTT. After centrifuging at 25 000g for 60 min, the supernatant underwent a reductive alkylation reaction. The concentration of the protein solution was determined using the Bradford method with BSA as a standard. For protein digestion, 100 μg of protein sample was digested with trypsin at 20 : 1 (w/w) for 4 h at 37 °C and then digested 8 h at 37 °C after adding the same amount trypsin. Sample digestions were vacuum dried and reconstituted with 0.5 M TEAB. iTRAQ labeling was performed using iTRAQ 8-plex kits (AB Sciex, USA) according to the manufacturer's protocol.^{42 (link)}

Wang R., Lu C., Shu Z., Yuan X., Jiang H, & Guo H. (2020). iTRAQ-based proteomic analysis reveals several key metabolic pathways associated with male sterility in Salvia miltiorrhiza. RSC Advances, 10(29), 16959-16970.

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Quantitative Protein Profiling by iTRAQ

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The protein profiling by iTRAQ was performed by the BGI. Total protein (100 μg) of each sample solution was digested with Trypsin Gold (Promega) at 37 °C for 16 h. After digestion, peptides were freeze-dried by vacuum centrifugation. Peptides of eight samples in a biological replicate were reconstituted in 0.5 M TEAB and then labeled with iTRAQ 8-plex kits according to the manufacturer’s manual (AB Sciex Inc.) as follows: Ch₅, 113; Tr₅, 114; Ch₁₀, 115; Tr₁₀, 116; Ch₁₅, 117; Tr₁₅, 118; Ch₂₀, 119; and Tr₂₀, 121. The labeled peptides in a biological replicate were pooled as a set and resolved into 12 fractions using an Ultremex SCX column (Phenomenex) in an LC-20AB HPLC system (Shimadzu). The eluted fractions were then desalted using a Strata X C18 column (Phenomenex) and dried under vacuum.

Lin Z., Wang Z., Zhang X., Liu Z., Li G., Wang S, & Ding Y. (2017). Complementary Proteome and Transcriptome Profiling in Developing Grains of a Notched-Belly Rice Mutant Reveals Key Pathways Involved in Chalkiness Formation. Plant and Cell Physiology, 58(3), 560-573.

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Protein Digestion and Peptide Labeling

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Protein solutions (100 µg) were diluted 4-times with 100 mM tetraethylammonium bromide, after which proteins were digested overnight with Trypsin Gold (Promega, Madison, WI, USA) at 37 °C (40:1, protein:trypsin). The peptides were desalted with a Strata X C18 column (Phenomenex, Torrance, CA, USA) and then vacuum-dried according to the protocol recommended by the manufacturer. Peptide samples were labelled using iTRAQ 8-plex kits (AB Sciex Inc., MA, USA). The two ‘Tunisia’ samples (TP1 and TP2) were labelled with iTRAQ tags 113 and 115, while the two ‘White’ samples (SP1 and SP2) were labelled with tags 117 and 119.

Luo X., Cao D., Li H., Zhao D., Xue H., Niu J., Chen L., Zhang F, & Cao S. (2018). Complementary iTRAQ-based proteomic and RNA sequencing-based transcriptomic analyses reveal a complex network regulating pomegranate (Punica granatum L.) fruit peel colour. Scientific Reports, 8, 12362.

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Quantitative Proteomics of Plant Stress

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iTRAQ analysis was implemented at BGI(Shenzhen, China). Total proteins were extracted from the roots and leaves tissue of CCRI-79 plants using a phenol extraction procedure [30 (link),31 (link)]. Two biological replicates were carried out for each sample.
Protein concentrations were determined using the Bradford method [32 ]. An equal amount of protein was prepared for each biological replicate. Protein samples from the roots and leaves were reduced with 10 mM DTT, alkylated with 55 mM iodoacetamide, digested using sequencing grade trypsin (Promega) at a ratio of 1:10 (w:w) for12 h at 37°C, and labeled using iTRAQ 8-plex kits (AB Sciex Inc., MA, USA) according to manufacturer’s manual. The root samples were labeled with iTRAQ tags 113 and114 (CK), 117 and 118 (salt treatment). The leaf samples were labeled with tags 115 and116 (CK), 119 and 120 (salt treatment).

Chen T., Zhang L., Shang H., Liu S., Peng J., Gong W., Shi Y., Zhang S., Li J., Gong J., Ge Q., Liu A., Ma H., Zhao X, & Yuan Y. (2016). iTRAQ-Based Quantitative Proteomic Analysis of Cotton Roots and Leaves Reveals Pathways Associated with Salt Stress. PLoS ONE, 11(2), e0148487.

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Quantitative Analysis of Salivary Proteins

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Equal amounts of protein (50 μg each) from each saliva sample were precipitated with 6 volumes of chilled acetone at −20°C overnight, followed by centrifugation for 10 min at 5000 RCF. Subsequently, proteins were denatured, reduced, digested and labelled following the protocol recommended by AB Sciex. Briefly, the pellet was resuspended in 20 μL of dissolution buffer containing 1 μL of denaturant followed by 2 μL of reducing agent. Samples were incubated for 1 h at 60°C. After centrifugation, 1 μL of cysteine blocking reagent was added and samples were incubated for additional 10 min at room temperature (RT). Proteins were digested with trypsin (AB Sciex) and incubated overnight at 37°C. The peptides resulting from trypsin digestion of salivary proteins from 8 different samples were each labeled with a different isobaric tag (113–121) from an iTRAQ 8-plex kit (AB Sciex) and combined.

Costa-da-Silva A.C., Aure M.H., Dodge J., Martin D., Dhamala S., Cho M., Rose J.J., Bassim C.W., Ambatipudi K., Hakim F.T., Pavletic S.Z, & Mays J.W. (2021). Salivary ZG16B expression loss follows exocrine gland dysfunction related to oral chronic graft-versus-host disease. iScience, 25(1), 103592.

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Comparative Protein Profiling of Homo Sapiens

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The PRM was performed by PTM BIO (Hangzhou, China). Briefly, the differential expression of selected proteins was detected using a Q-exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). After protein extraction and trypsin digestion, the tryptic peptides were prepared using an EASY-nLC 1000 UPLC system. And the resulting MS data were processed using Skyline software (v.3.6). The iTraq was performed by Luming Biotechnology (Shanghai, China). Briefly, peptides were labeled using an iTraq 8-plex kit (AB SCIEX, Washington, DC) in two technical replicates as per manufacturer’s instructions. The iTraq-based LC-MS/MS analysis was carried out using a Triple TOF 5600 System (AB SCIEX, USA) fitted with a Nanospray III source (AB SCIEX, USA) and a pulled quartz tip as the emitter (New Objectives, USA). Data were processed by the protein pilot software (v. 5.0, AB SCIEX, USA) against Homo Sapiens database using the Paragon algorithm.

Zhu G., Wang F., Li H., Zhang X., Wu Q., Liu Y., Qian M., Guo S., Yang Y., Xue X., Sun F., Qiao Y, & Pan Q. (2021). N-Myristoylation by NMT1 Is POTEE-Dependent to Stimulate Liver Tumorigenesis via Differentially Regulating Ubiquitination of Targets. Frontiers in Oncology, 11, 681366.

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iTRAQ Peptide Labeling Protocol

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The dried peptide was reconstituted in 20 μL 0.5 M TEAB and processed by following the manufacturer's protocol (iTRAQ 8plex kit, AB Sciex). Briefly, iTRAQ reagent was thawed and reconstituted in 70 μL acetonitrile. Peptide and reagent were mixed and incubated at room temperature for 1 hour and then dried by vacuum centrifugation.

Xue Z., Li A., Zhang X., Yu W., Wang J., Zhang Y., Gao X, & Kou X. (2019). iTRAQ based proteomic analysis of PM2.5 induced lung damage. RSC Advances, 9(21), 11707-11717.

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Proteomics analysis of exosome-treated cells

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LX-2 cells were plated into 60 mm dishes and cultured with HCT116-derived exosomes for 48 hours. Then the cells were collected for proteomics analysis. Isobaric tags for relative and absolute quanti cation (iTRAQ)-based proteomic analysis was conducted by Omicsolution Co., Ltd (Shanghai, China). Brie y, the cell lysate was extracted from each sample, and a total of 100 μg of each sample were removed and labeled using an iTRAQ-8plex kit (AB SCIEX, USA) according to the manufacturer's instructions. Each set of labeled peptides was pooled and resuspended in strong cation exchange (SCX) prefractionation using SCX chromatography on an Ultremex SCX column (4.6 × 250 mm) with a 3000 high-performance liquid chromatography system (Thermo Fisher DINOEX, Waltham, MA, USA). Each fraction was analyzed using nano liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS); nano LC-MS was performed using a TripleTOF ® 5600 mass spectrometer for MS/MS (AB SCIEX) interfaced with Eksigent nano LC system. The functions of the multiple protein contents were obtained by searching the UniProt database. Proteins were ltered by using the following thresholds: p value < 0.05 and 1.2-fold change. All differentially expressed proteins were selected for heat map and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis.

Zhang C., Wang X., Zhang P., He T., Han J., Zhang R., Lin J., Fan J., Lu L., Zhu W., Jia H., Zhang J., & Chen J. (2021). Cancer-Secreted Exosomal HSPC111 Promotes Colorectal Cancer Liver Metastasis By Reprogramming Lipid Metabolism in Cancer-Associated Fibroblasts.

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