RIMVECs were treated with IL-1α (final concentration of 10 ng/mL) at 37°C incubated in a 5% CO2 atmosphere for 0, 2, 4, and 8 h. The proteins of RIMVCEs were extracted by RIPA lysate buffer (Beyotime) and quantified by a BSA kit (Beyotime). The concentrations of sample proteins were detailed in Supplementary Table S1 . Two hundred microgram proteins were incubated in iTRAQ-4-plex kit (AB Sciex, PN: 4352135) proteolysis. The iTRAQ labeled, LC-MS/MS analysis and MALDI-TOF-TOF identifications were conducted by BIOMS company. Labels of 114, 115, 116, and 117 are represented 2, 4, 6, and 0 h treatment, respectively (Supplementary Table S1 ).
Itraq 4 plex kit
Manufactured by AB Sciex
Sourced in United States
The iTRAQ 4-plex kit is a quantitative proteomics tool designed to enable the simultaneous identification and relative quantification of proteins from up to four different samples. It utilizes isobaric tags that are covalently attached to peptides derived from protein samples, allowing for the multiplexed analysis of complex protein mixtures.
Automatically generated - may contain errors
Lab products found in correlation
C18 desalting column, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Ripa lysate buffer, by Beyotime (1 mentions)
Lithium dodecyl sulfate (lds), by Thermo Fisher Scientific (1 mentions)
Trypsin, by AB Sciex (1 mentions)
Cysteine blocking reagent, by AB Sciex (1 mentions)
Reducing reagent, by AB Sciex (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
Sep pak c18 cartridge, by Waters Corporation (1 mentions)
C18 spe cartridge, by Agilent Technologies (1 mentions)
Offgel fractionator, by Agilent Technologies (1 mentions)
Strata x c18 spe column, by Phenomenex (1 mentions)
10 protocols using itraq 4 plex kit
1
Quantifying Temporal Proteome Changes in RIMVECs
2
Proteomic Analysis of Brain Samples
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The animals were euthanatized and quickly decapitated to remove the brain. The whole brains were homogenized in lysis buffer [50 mM ammonium bicarbonate (pH 8.0), 0.1% SDS with protease inhibitor cocktail from (SIGMA)] and for further efficient disruption and homogenization of tissue, a mild sonication was done using Bioruptor. The obtained lysates were cold-centrifuged at 14000 g for 15 minutes, and the protein concentration was quantified in collected supernatant using Barford assay with BSA as a standard. Further protein sample was cleaned up by acetone precipitation. For each group 80 µg of protein was taken and six volume of chilled acetone was added for precipitation and samples were air dried after decanting acetone.
Prior to trypsin digestion all the protein samples were gone through reduction and cysteine blocking using the reagents provided with iTRAQ 4-plex kit (AB SCIEX). Digestion and labelling of proteins were done according to manufacturer's protocol. Subsequently all the labelled samples were pooled, vacuum dried and further cleaned up using C18 desalting column (Pierce Thermoscientific). The final fraction again concentrated using vacuum concentrator and reconstituted with 10 µl of 0.1% formic acid for LC-MS/MS analysis.
Prior to trypsin digestion all the protein samples were gone through reduction and cysteine blocking using the reagents provided with iTRAQ 4-plex kit (AB SCIEX). Digestion and labelling of proteins were done according to manufacturer's protocol. Subsequently all the labelled samples were pooled, vacuum dried and further cleaned up using C18 desalting column (Pierce Thermoscientific). The final fraction again concentrated using vacuum concentrator and reconstituted with 10 µl of 0.1% formic acid for LC-MS/MS analysis.
3
Quantitative Protein Profiling by iTRAQ
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The biological duplicate protein samples (0.75 mg) were reconstituted in 150 µL 1 × LDS (Invitrogen) with 50 mM DTT (dithiothreitol), followed by incubation at 90 °C for 10 min. Ten percent of each sample were loaded and run into SDS-PAGE gel. The in-gel protein samples were reduced with 10 mM DTT at 60 °C for 30 min and alkylated with 20 mM iodoacetamide at room temperature and dark conditions for 1 h. The samples were digested overnight with trypsin (1/50, w/w, trypsin/sample) at 37 °C. The digested peptides were subsequently extracted from the SDS-PAGE gel with buffer (60% acetonitrile, 5% formic acid) and vacuum-dried to remove residual amine from the reagents. Peptides were labeled with iTRAQ 4-plex Kit (ABSciex, USA) according to the manufacturer’s protocol and then combined after labeling.
4
Protein Extraction and Quantification for Comparative Proteomics
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Protein extraction, digestion, iTRAQ labeling, and mass spectrometry were conducted using protocols from previous paper (Xing et al. 2014 (link)). Briefly, total protein was extracted from each sample using Plant Total Protein Extraction Kit PE0230 (Sigma-Aldrich, USA) according to the manufacturer’s instructions. Bradford method was selected to determine the protein content. Proteins of each sample were detected by 10% SDS-PAGE (Fig. 1 ). Each of the samples (75 μg in total) was deoxidized with 20 mM DTT, alkylated with 50 mM IAA and digested with trypsin.
The peptide samples were labeled using the iTRAQ 4-plex kit (AB sciex, USA) according to the manufacturer’s protocol. Then, EP and LP were labeled by 116 and 117 iTRAQ, respectively. After labeling, the equal amounts of each sample were mixed together and lyophilized.
The peptide samples were labeled using the iTRAQ 4-plex kit (AB sciex, USA) according to the manufacturer’s protocol. Then, EP and LP were labeled by 116 and 117 iTRAQ, respectively. After labeling, the equal amounts of each sample were mixed together and lyophilized.
5
Proteomic Analysis of Brain Samples
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The animals were euthanatized and quickly decapitated to remove the brain. The whole brains were homogenized in lysis buffer [50 mM ammonium bicarbonate (pH 8.0), 0.1% SDS with protease inhibitor cocktail from (SIGMA)] and for further efficient disruption and homogenization of tissue, a mild sonication was done using Bioruptor. The obtained lysates were cold-centrifuged at 14000 g for 15 minutes, and the protein concentration was quantified in collected supernatant using Barford assay with BSA as a standard. Further protein sample was cleaned up by acetone precipitation. For each group 80 µg of protein was taken and six volume of chilled acetone was added for precipitation and samples were air dried after decanting acetone.
Prior to trypsin digestion all the protein samples were gone through reduction and cysteine blocking using the reagents provided with iTRAQ 4-plex kit (AB SCIEX). Digestion and labelling of proteins were done according to manufacturer's protocol. Subsequently all the labelled samples were pooled, vacuum dried and further cleaned up using C18 desalting column (Pierce Thermoscientific). The final fraction again concentrated using vacuum concentrator and reconstituted with 10 µl of 0.1% formic acid for LC-MS/MS analysis.
Prior to trypsin digestion all the protein samples were gone through reduction and cysteine blocking using the reagents provided with iTRAQ 4-plex kit (AB SCIEX). Digestion and labelling of proteins were done according to manufacturer's protocol. Subsequently all the labelled samples were pooled, vacuum dried and further cleaned up using C18 desalting column (Pierce Thermoscientific). The final fraction again concentrated using vacuum concentrator and reconstituted with 10 µl of 0.1% formic acid for LC-MS/MS analysis.
6
Protein Labeling and Quantification Using iTRAQ
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Extracted proteins (200 μg) were transferred into new tubes and reacted with 4 μL of reducing reagent (AB Sciex Inc., Framingham, MA) for 1 h at 60°C. Then, 2 μL of cysteine-blocking reagent (AB Sciex Inc.) was added to the sample, followed by incubation at room temperature for 10 min. The reductive alkylated proteins were incubated with sequencing-grade modified trypsin (Promega, Madison, WI) at a ratio of 50:1 (protein:trypsin) at 37°C for 16 h.
Digested protein samples were labeled using an iTRAQ 4-plex kit (AB Sciex Inc.) according to the manufacturer's instructions with some modification. Briefly, 1 vial of 4-plex iTRAQ labeling reagent was used for each protein sample. The iTRAQ reagent was solubilized in isopropanol and then added to the protein sample to a final organic concentration of at least 65% (vol/vol). After 2 h of iTRAQ labeling, the reaction was quenched by addition of 100 μL of deionized water. The samples were then mixed at equal ratios and dried by centrifugal evaporation.
Digested protein samples were labeled using an iTRAQ 4-plex kit (AB Sciex Inc.) according to the manufacturer's instructions with some modification. Briefly, 1 vial of 4-plex iTRAQ labeling reagent was used for each protein sample. The iTRAQ reagent was solubilized in isopropanol and then added to the protein sample to a final organic concentration of at least 65% (vol/vol). After 2 h of iTRAQ labeling, the reaction was quenched by addition of 100 μL of deionized water. The samples were then mixed at equal ratios and dried by centrifugal evaporation.
7
Quantitative Proteomic Analysis via iTRAQ
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In each iTRAQ experiment, the resulting peptides of tryptic digestion were labeled using iTRAQ 4-plex kit (AB SCIEX, Foster City, CA, USA) according to the manufacturer's instructions as follows: STD-C, tag-114; STD-FOM, tag-115; STD-GSE, tag-116 and STD-FOM&GSE, tag-117, for the first STD experiment; and HFHS-C, tag-114; HFHS-FOM, tag-115; HFHS-GSE, tag-116 and HFHS-FOM&GSE, tag-117, for the second HFHS experiment (Table 1).
After labeling, the samples were pooled, dried and desalted using a SEP-PAK C18 Cartridge (Waters). Finally cleaned tryptic peptides were evaporated to dryness and stored at -20°C for further analysis.
After labeling, the samples were pooled, dried and desalted using a SEP-PAK C18 Cartridge (Waters). Finally cleaned tryptic peptides were evaporated to dryness and stored at -20°C for further analysis.
8
Multiplexed Proteomic Analysis of BAL Fluid
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We performed an isobaric tagging for accurate quantitation (iTRAQ) method for multiplexed proteomic analysis of BAL fluid from infected mice. For this, BAL fluid samples from 3mice each at Days 0, 5, 14 and 21 were concentrated and the protein concentration was measured in each sample using the BCA method in a kit (Pierce chemical co. USA). Seventy-five micrograms of protein was subjected with trypsin digestion at a ratio of 1∶80 (trypsin: protein) followed by labeling with isobaric tags using the 4-plex-iTRAQ kit (AB Sciex Pte Ltd, USA) as per manufacturer's instructions. The peptides from Day-5 were labeled with 115 reporter ions while those from Days 14 and 21 were labeled with 116 and 117 reported ions. Peptides from Day-0 were labeled with 114 reporter ion which served as a reference control. The individually labeled peptide samples from each group were pooled and multiplexed peptide samples were desalted using a C-18 SPE cartridge (Agilent technologies), resolved by isoelectric focusing on a pH3–10 strip (GE healthcare) on an OFFGEL fractionator (Agilent technologies). The resolved peptides were collected in 12 fractions, dried and dissolved in 15 µl of 2%ACN/0.1%TFA.
9
Comparative Proteomic Analysis of WT and APKO Dendritic Cells
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Three samples from the WT-DEN and APKO-DEN groups were selected for proteomic analysis. ITRAQ (isolated tag for relative and absolute quantification) proteomic technology was used to identify differential proteins. The sample protein was extracted and evaluated by SDS-PAGE. The sample was digested with the filter-aided proteome preparation (FASP) method and labeled with a 4-plex iTRAQ Kit (AB SCIEX). The peptide segment was preseparated by high pH reversed-phase liquid chromatography, and using a C18 column. After preseparation, the partial supernatant of the sample was analyzed by liquid chromatography-mass spectrometry (Thermo™ Q Exactive HF type). The parameters were searched by Proteome Discovery.
10
Protein Reduction, Alkylation, and Trypsin Digestion for iTRAQ Labeling
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For protein digestion, 10 mM DTT and 20 mM IAA were used to reduce and alkylate the protein solution at 37 °C for 1 h and at 25 °C for 45 min, respectively. For trypsin digestion, 100 mM TEAB (Triethylammonium bicarbonate) (Sigma-Aldrich, Darmstadt, Germany) was added to the protein sample until the urea concentration was less than 2 M. Trypsin was then added (w/w, protein:trypsin ratio of 50:1) and the resulting solution was held overnight. A second digestion was performed for 4 h in the ratio of 100:1 (w/w, protein:trypsin).
The tryptic peptides were desalted by Strata X C18 SPE column (Phenomenex), vacuum-dried, dissolved in 0.5 M TEAB buffer and processed according to the manufacturer’s instructions for 4-plex iTRAQ kit (AB SCIEX). Briefly, one unit of iTRAQ reagent was thawed and reconstituted in 24 μL acetonitrile (ACN). A total of 12 samples (3 biological replicates) were iTRAQ labeled. The peptide mixtures were labeled with respective isobaric tags (114 for S1 (COSMA); 115 for S2 (IADA); 116 for S3 (COSMN); 117 for S4 (IADN)), then incubated at 25 °C for 2 h and pooled, desalted and dried by vacuum centrifugation. Three biological replicates were performed.
The tryptic peptides were desalted by Strata X C18 SPE column (Phenomenex), vacuum-dried, dissolved in 0.5 M TEAB buffer and processed according to the manufacturer’s instructions for 4-plex iTRAQ kit (AB SCIEX). Briefly, one unit of iTRAQ reagent was thawed and reconstituted in 24 μL acetonitrile (ACN). A total of 12 samples (3 biological replicates) were iTRAQ labeled. The peptide mixtures were labeled with respective isobaric tags (114 for S1 (COSMA); 115 for S2 (IADA); 116 for S3 (COSMN); 117 for S4 (IADN)), then incubated at 25 °C for 2 h and pooled, desalted and dried by vacuum centrifugation. Three biological replicates were performed.
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