The iTRAQ™ Reagents Kit was from Applied Biosystems (USA). TSPY1 small hairpin (sh) RNA fragments were purchased from Genechem (Shanghai, China). Rabbit polyclonal to TSPY1 was from Abcam Company. Mouse monoclonal to Flag was purchased from Sigma. Taq polymerase purchased from TAKARA. Lipofectamine 2000 was purchased from Invitrogen. Dulbecco’s modified Eagle’s (DMEM) medium, Roswell Park Memorial Institute 1640(RPMI-1640) and fetal bovine serum (FBS) were from Sigma Group and Gibco Company.
Itraq reagents kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ITRAQ™ Reagents Kit is a set of chemical reagents designed for quantitative proteomic analysis. The kit enables the labeling and identification of peptides in complex samples, allowing for the comparison of protein abundance across multiple conditions.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Taq polymerase, by Takara Bio (1 mentions)
Mouse monoclonal to flag, by Merck Group (1 mentions)
Sep pak c18, by Waters Corporation (1 mentions)
Sequence grade modified trypsin, by Promega (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Acetone, by Merck Group (1 mentions)
Sodium citrate buffer, by Merck Group (1 mentions)
Polysulfoethyl column, by Nest Group (1 mentions)
Rabbit polyclonal antibodies, by Abcam (1 mentions)
Formic acid, by Merck Group (1 mentions)
Trypsin, by Promega (1 mentions)
4 protocols using itraq reagents kit
1
iTRAQ-based Protein Quantification with TSPY1 Knockdown
2
Protein Quantification and Labeling for Mass Spectrometry
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We determined the protein concentration of the supernatants using the bicinchoninic acid protein assay, and then transferred 100 μg protein per condition into new tubes and adjusted each to a final volume of 10 μL with 100 mM triethylammonium bicarbonate (TEAB). To this 5 μL of 200 mM DL-dithiothreitol were added and incubated at 55 °C for 1 h, then 5 μL of the 375 mM iodoacetamide was added to the sample and incubated for 30 min protected from light at room temperature.
For each sample, proteins were precipitated with ice-cold acetone, and then re-dissolved in 20 μL TEAB. Proteins were then digested with sequence-grade modified trypsin (Promega, Madison, WI), and the resultant peptide mixture was labeled using chemicals from the iTRAQ reagents kit (Applied Biosystems, Foster City, CA). The labeled samples were combined, desalted using a C18 SPE column (Sep-Pak C18, Waters, Milford, MA) and dried under vacuum.
For each sample, proteins were precipitated with ice-cold acetone, and then re-dissolved in 20 μL TEAB. Proteins were then digested with sequence-grade modified trypsin (Promega, Madison, WI), and the resultant peptide mixture was labeled using chemicals from the iTRAQ reagents kit (Applied Biosystems, Foster City, CA). The labeled samples were combined, desalted using a C18 SPE column (Sep-Pak C18, Waters, Milford, MA) and dried under vacuum.
3
iTRAQ-Based Proteomics Workflow
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iTRAQ™ Reagents Kit was bought from Applied Biosystems (San Jose, CA, USA). The acetonitrile, formic acid, acetone, trypsilin, and sodium citrate buffer were from Sigma-Aldrich (California, CA, USA). The Zorbax 300SB-C18 reversed-phase column (Microm, Auburn, CA, USA), the Polysulfoethyl column (The Nest Group, Southborough, MA, USA) and QSTAR XL System (Applied Biosystem, California, CA, USA) were for 2D LC-MS/MS. Sep-Pak Vac C18 cartridges was obtained from Millipore Corporation (Minneapolis, Minnesota, USA). The rabbit polyclonal antibodies were purchased from Abcam (London, UK).
4
Protein Extraction and Quantification
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Briefly, 0.5 g leaf samples were homogenized in 2 mL lysis buffer, including 8 M Urea, 50 mM Tris-HCl (pH 8), and 0.2% SDS sonicated for five minutes on ice. The samples were then centrifuged at 12,000× g at 4 °C for 15 min, and the supernatant was moved to a new tube. The concentration of protein was determined using a Bradford protein assay. Extracts from each sample were reduced with 2 mM DTT for 1 h and alkylated with sufficient iodoacetic acetate for 1 h in the dark at room temperature. A 4-fold volume of precooled acetone was mixed with the samples and incubated at −20 °C for 1 h; the samples were then centrifuged to collect precipitation. After they were washed three times with cold acetone, the pellets were dissolved in lysis buffer containing 0.1 M triethyl ammonium bicarbonate (TEAB, pH 8.5) and 8 M urea. An equal number of proteins were digested with trypsin (Promega, Madison, WI, USA) at a ratio of 1:50 (w:w) for 16 h at 37 °C and iTRAQ reagents kit (Applied Biosystems, Framingham, MA, USA) were used to label 100 micrograms of digested proteins according to the manufacturer’s protocol.
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