itraq reagents multiplex kit  (Millipore)


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    Name:
    iTRAQ Reagents Multiplex Kit
    Description:
    Contains iTRAQ R reagents 114 115 116 117 the appropriate buffers and reagents for ten 2 plex six 3 plex or five 4 plex assays 10 PK trypsin is sold separately Each individual reagent is capable of labeling up to 100 mug of protein
    Catalog Number:
    4352135
    Price:
    None
    Applications:
    The iTRAQ(R) Reagents Multiplex Kit provides iTRAQ(R) reagents and protocol to label peptides after digestion. This widely used generic labeling kit can be used for virtually any workflow.iTRAQ(R) Reagents Multiplex Kit has been used for the labeling of peptides for LC-MS (liquid chromatography-mass spectrometry)/MS analysis.
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    Structured Review

    Millipore itraq reagents multiplex kit
    Schematic representation of the integrated <t>BONCAT-iTRAQ</t> 4-plex workflow. Parasites maintained in methionine-free medium for 30 minutes (step 1) were subjected to starvation in DPBS for 1, 2 and 3 hour durations, and the NSPs in the parasites were metabolically labelled with AHA treatment or DMSO treatment as control (step 2). The parasites were then lysed, and the NSPs were chemically tagged using bio-orthogonal click reaction with a Biotin-Alkyne (step 3). The biotin-tagged proteins were affinity enriched on <t>NeutrAvidin</t> beads, and following on-bead tryptic digestion (step 4), the released peptides were subjected to iTRAQ 4-plex labelling (step 5). iTRAQ channel 114 was used for labelling the DMSO control sample, whilst channels 115, 116 and 117 were used respectively for labelling the NSPs at 1 hour, 2 hour and 3 hour starvation. The samples after pooling together (step 6) were analysed by nanoLC-MS/MS (step 7). The relative intensity values of the reporter ions (iTRAQ ions 114, 115, 116 and 117) in the tandem mass spectra (MS/MS) of each detected tryptic peptide provide estimation of the relative abundance of the peptide in the corresponding sample.
    Contains iTRAQ R reagents 114 115 116 117 the appropriate buffers and reagents for ten 2 plex six 3 plex or five 4 plex assays 10 PK trypsin is sold separately Each individual reagent is capable of labeling up to 100 mug of protein
    https://www.bioz.com/result/itraq reagents multiplex kit/product/Millipore
    Average 99 stars, based on 2598 article reviews
    Price from $9.99 to $1999.99
    itraq reagents multiplex kit - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "A BONCAT-iTRAQ method enables temporally resolved quantitative profiling of newly synthesised proteins in Leishmania mexicana parasites during starvation"

    Article Title: A BONCAT-iTRAQ method enables temporally resolved quantitative profiling of newly synthesised proteins in Leishmania mexicana parasites during starvation

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0007651

    Schematic representation of the integrated BONCAT-iTRAQ 4-plex workflow. Parasites maintained in methionine-free medium for 30 minutes (step 1) were subjected to starvation in DPBS for 1, 2 and 3 hour durations, and the NSPs in the parasites were metabolically labelled with AHA treatment or DMSO treatment as control (step 2). The parasites were then lysed, and the NSPs were chemically tagged using bio-orthogonal click reaction with a Biotin-Alkyne (step 3). The biotin-tagged proteins were affinity enriched on NeutrAvidin beads, and following on-bead tryptic digestion (step 4), the released peptides were subjected to iTRAQ 4-plex labelling (step 5). iTRAQ channel 114 was used for labelling the DMSO control sample, whilst channels 115, 116 and 117 were used respectively for labelling the NSPs at 1 hour, 2 hour and 3 hour starvation. The samples after pooling together (step 6) were analysed by nanoLC-MS/MS (step 7). The relative intensity values of the reporter ions (iTRAQ ions 114, 115, 116 and 117) in the tandem mass spectra (MS/MS) of each detected tryptic peptide provide estimation of the relative abundance of the peptide in the corresponding sample.
    Figure Legend Snippet: Schematic representation of the integrated BONCAT-iTRAQ 4-plex workflow. Parasites maintained in methionine-free medium for 30 minutes (step 1) were subjected to starvation in DPBS for 1, 2 and 3 hour durations, and the NSPs in the parasites were metabolically labelled with AHA treatment or DMSO treatment as control (step 2). The parasites were then lysed, and the NSPs were chemically tagged using bio-orthogonal click reaction with a Biotin-Alkyne (step 3). The biotin-tagged proteins were affinity enriched on NeutrAvidin beads, and following on-bead tryptic digestion (step 4), the released peptides were subjected to iTRAQ 4-plex labelling (step 5). iTRAQ channel 114 was used for labelling the DMSO control sample, whilst channels 115, 116 and 117 were used respectively for labelling the NSPs at 1 hour, 2 hour and 3 hour starvation. The samples after pooling together (step 6) were analysed by nanoLC-MS/MS (step 7). The relative intensity values of the reporter ions (iTRAQ ions 114, 115, 116 and 117) in the tandem mass spectra (MS/MS) of each detected tryptic peptide provide estimation of the relative abundance of the peptide in the corresponding sample.

    Techniques Used: Metabolic Labelling, Mass Spectrometry

    Schematic representation of BONCAT-iTRAQ 3-plex workflow for the identification of starvation-responsive NSPs. Parasites maintained in methionine-free medium for 30 minutes (step 1) were subjected to starvation in DPBS for 1 hour and 2 hour durations and the NSPs in the parasites were metabolically labelled with AHA treatment (step 2). Parallel AHA treatment on non-starved parasites were performed as controls. The parasites were then lysed, and the NSPs from the starved and non-starved batches were chemically tagged using click reaction with Biotin-Alkyne (step 3). The biotin-tagged proteins were affinity enriched on NeutrAvidin beads and following on-bead tryptic digestion (step 4), the released peptides were subjected to iTRAQ 3-plex labelling (step 5). iTRAQ channel 114 was used for labelling the AHA-treated non-starved control sample, whilst channels 116, and 117 were used respectively for labelling the NSPs at 1 hour and 2 hour durations of starvation. The samples after pooling together (step 6) were analysed by LC-MS/MS (step 7).
    Figure Legend Snippet: Schematic representation of BONCAT-iTRAQ 3-plex workflow for the identification of starvation-responsive NSPs. Parasites maintained in methionine-free medium for 30 minutes (step 1) were subjected to starvation in DPBS for 1 hour and 2 hour durations and the NSPs in the parasites were metabolically labelled with AHA treatment (step 2). Parallel AHA treatment on non-starved parasites were performed as controls. The parasites were then lysed, and the NSPs from the starved and non-starved batches were chemically tagged using click reaction with Biotin-Alkyne (step 3). The biotin-tagged proteins were affinity enriched on NeutrAvidin beads and following on-bead tryptic digestion (step 4), the released peptides were subjected to iTRAQ 3-plex labelling (step 5). iTRAQ channel 114 was used for labelling the AHA-treated non-starved control sample, whilst channels 116, and 117 were used respectively for labelling the NSPs at 1 hour and 2 hour durations of starvation. The samples after pooling together (step 6) were analysed by LC-MS/MS (step 7).

    Techniques Used: Metabolic Labelling, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    2) Product Images from "A BONCAT-iTRAQ method enables temporally resolved quantitative profiling of newly synthesised proteins in Leishmania mexicana parasites during starvation"

    Article Title: A BONCAT-iTRAQ method enables temporally resolved quantitative profiling of newly synthesised proteins in Leishmania mexicana parasites during starvation

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0007651

    BONCAT in L . mexicana promastigotes under starvation. Promastigotes were cultured in methionine-free Schneider’s medium (30 minutes) prior to incubation in DPBS for different time periods (1 hour to 7 hours). The starved parasites were treated with AHA (50μM; lanes 3 to 9) or DMSO (control; lane 2) with (lane 9) or without CHX (10μM) for the last 1 hour of starvation and the NSPs were profiled by in-gel fluorescence scanning following click chemistry with a TAMRA-Alkyne. A Coomassie blue staining of the same gel that demonstrates even loading across the gel lanes is shown on the right panel.
    Figure Legend Snippet: BONCAT in L . mexicana promastigotes under starvation. Promastigotes were cultured in methionine-free Schneider’s medium (30 minutes) prior to incubation in DPBS for different time periods (1 hour to 7 hours). The starved parasites were treated with AHA (50μM; lanes 3 to 9) or DMSO (control; lane 2) with (lane 9) or without CHX (10μM) for the last 1 hour of starvation and the NSPs were profiled by in-gel fluorescence scanning following click chemistry with a TAMRA-Alkyne. A Coomassie blue staining of the same gel that demonstrates even loading across the gel lanes is shown on the right panel.

    Techniques Used: Cell Culture, Incubation, Fluorescence, Staining

    3) Product Images from "Amelogenin-chitosan matrix promotes assembly of an enamel-like layer with a dense interface"

    Article Title: Amelogenin-chitosan matrix promotes assembly of an enamel-like layer with a dense interface

    Journal: Acta biomaterialia

    doi: 10.1016/j.actbio.2013.04.004

    a) Hardness and elastic modulus of healthy enamel, etched enamel, and reconstructed enamel repaired by chitosan hydrogel with and without amelogenin. * p ≤0.05 when compared with artificial caries; *** p
    Figure Legend Snippet: a) Hardness and elastic modulus of healthy enamel, etched enamel, and reconstructed enamel repaired by chitosan hydrogel with and without amelogenin. * p ≤0.05 when compared with artificial caries; *** p

    Techniques Used:

    a) TEM image of the original CS-AMEL hydrogel showing the elongated nanochain-like structure (white arrows). b) Cross-section SEM image of repaired layer after remineralization in amelogenin-chitosan gel for 3 days fused to the surface of the natural
    Figure Legend Snippet: a) TEM image of the original CS-AMEL hydrogel showing the elongated nanochain-like structure (white arrows). b) Cross-section SEM image of repaired layer after remineralization in amelogenin-chitosan gel for 3 days fused to the surface of the natural

    Techniques Used: Transmission Electron Microscopy

    SEM images of newly grown layer without amelogenin after remineralization in an artificial saliva solution for 7 days. a, c) top view, b, d) side view. a, b) Without, c, d) with chitosan gel.
    Figure Legend Snippet: SEM images of newly grown layer without amelogenin after remineralization in an artificial saliva solution for 7 days. a, c) top view, b, d) side view. a, b) Without, c, d) with chitosan gel.

    Techniques Used:

    SEM and TEM images of natural enamel and newly-grown layer after remineralization in amelogenin-chitosan gel for 7 days. a) Microstructure of native enamel. Black arrows indicate the crystallographic orientations of the apatite crystallites in native
    Figure Legend Snippet: SEM and TEM images of natural enamel and newly-grown layer after remineralization in amelogenin-chitosan gel for 7 days. a) Microstructure of native enamel. Black arrows indicate the crystallographic orientations of the apatite crystallites in native

    Techniques Used: Transmission Electron Microscopy

    a) XRD spectra of newly-grown layer after remineralization in a chitosan gel a) with and b) without amelogenin for 7 days. b) EDS spectrum of repaired layer after remineralization in a chitosan gel with amelogenin for 7 days.
    Figure Legend Snippet: a) XRD spectra of newly-grown layer after remineralization in a chitosan gel a) with and b) without amelogenin for 7 days. b) EDS spectrum of repaired layer after remineralization in a chitosan gel with amelogenin for 7 days.

    Techniques Used:

    SEM images of reconstructed enamel-like layers after ultrasonic treatment. a) backscattered electron image of the cross section, and b) second electron image of the surface of an ultrasonic-treated newly-grown layer obtained in a chitosan-amelogenin hydrogel.
    Figure Legend Snippet: SEM images of reconstructed enamel-like layers after ultrasonic treatment. a) backscattered electron image of the cross section, and b) second electron image of the surface of an ultrasonic-treated newly-grown layer obtained in a chitosan-amelogenin hydrogel.

    Techniques Used:

    CD and fluorescence spectra were measured with different mass ratio at pH a) 3.5, b) 5.5 and c) 8.0, revealing that the interaction between chitosan and amelogenin is pH dependent.
    Figure Legend Snippet: CD and fluorescence spectra were measured with different mass ratio at pH a) 3.5, b) 5.5 and c) 8.0, revealing that the interaction between chitosan and amelogenin is pH dependent.

    Techniques Used: Fluorescence

    4) Product Images from "Human microglia upregulate cytokine signatures and accelerate maturation of neural networks"

    Article Title: Human microglia upregulate cytokine signatures and accelerate maturation of neural networks

    Journal: bioRxiv

    doi: 10.1101/2020.03.24.006874

    Experimental models for microglia culture and xenotransplantation into brain tissue. (A) Primary prenatal microglia were purified with MACS-sorting and used for scRNAseq before and after in vitro culture. (B) UMAP of 16,876 MACS-purified microglia from GW23 that were acutely sequenced right after purification. (C) Clustering of microglia reveals molecularly heterogeneous subtypes. (D) Joint embedding and clustering of MACS-purified microglia and primary in silico -sorted microglia form nine unique clusters (E) Metadata annotation of the co-embedded object. Co-clustered MACS-purified and primary prenatal microglia demonstrate extensive overlap across all microglia subtypes in both samples. Bar plot represents the relative contribution of purified and acutely profiled microglia across clusters. (F) Purified primary human microglia can be cultured in two dimensions and expresses P2RY12 in vitro . (G) UMAP of 1,414 cultured primary microglia co-embedded with primary non-purified cells and clustered. (H) Relative contribution of cultured and acutely profiled microglia across clusters. (J) Xenotransplantation model of human microglia into adult brain tissue slice cultured ex vivo . Adult human slices were maintained in culture for two days and then MACS-purified microglia labeled with DiI were added to the slices. (K) Transplanted microglia (labelled with DiI) after 5 days in culture integrate into brain tissue. (L) Control nontransplanted tissue slices maintain microglia in culture.
    Figure Legend Snippet: Experimental models for microglia culture and xenotransplantation into brain tissue. (A) Primary prenatal microglia were purified with MACS-sorting and used for scRNAseq before and after in vitro culture. (B) UMAP of 16,876 MACS-purified microglia from GW23 that were acutely sequenced right after purification. (C) Clustering of microglia reveals molecularly heterogeneous subtypes. (D) Joint embedding and clustering of MACS-purified microglia and primary in silico -sorted microglia form nine unique clusters (E) Metadata annotation of the co-embedded object. Co-clustered MACS-purified and primary prenatal microglia demonstrate extensive overlap across all microglia subtypes in both samples. Bar plot represents the relative contribution of purified and acutely profiled microglia across clusters. (F) Purified primary human microglia can be cultured in two dimensions and expresses P2RY12 in vitro . (G) UMAP of 1,414 cultured primary microglia co-embedded with primary non-purified cells and clustered. (H) Relative contribution of cultured and acutely profiled microglia across clusters. (J) Xenotransplantation model of human microglia into adult brain tissue slice cultured ex vivo . Adult human slices were maintained in culture for two days and then MACS-purified microglia labeled with DiI were added to the slices. (K) Transplanted microglia (labelled with DiI) after 5 days in culture integrate into brain tissue. (L) Control nontransplanted tissue slices maintain microglia in culture.

    Techniques Used: Purification, Magnetic Cell Separation, In Vitro, In Silico, Cell Culture, Ex Vivo, Labeling

    Related Articles

    Multiplex Assay:

    Article Title: A BONCAT-iTRAQ method enables temporally resolved quantitative profiling of newly synthesised proteins in Leishmania mexicana parasites during starvation
    Article Snippet: .. Chemicals and reagents L-Azidohomoalanine (AHA; Iris Biotech GmbH), Cycloheximide (CHX; ACROS Organics), Tris (2-carboxyethy)phosphine hydrochloride (TCEP; Sigma Aldrich), 5-Tetramethylrhodamine-Alkyne (TAMRA-Alkyne; Jena Bioscience), Acetylene-PEG4-Biotin (Biotin-Alkyne; Jena Bioscience), Tris [(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA; Sigma Aldrich), Dimethyl sulfoxide (DMSO; Sigma Aldrich), Copper sulphate (CuSO4 ; Sigma Aldrich), 2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris; Sigma Aldrich), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES; Sigma Aldrich), Sodium chloride (NaCl; Fisher Scientific), Sodium dodecyl sulphate (SDS; Fisher Scientific), Sodium bicarbonate (NaHCO3 ; ACROS Organics), Calcium chloride (CaCl2 ; Sigma Aldrich), Urea (Sigma Aldrich), 1,4-Dithiothreitol (DTT; Sigma Aldrich), 2-Chloroacetamide (CAA; Sigma Aldrich), L-Glutamine solution (Sigma Aldrich), Benzonase (Sigma Aldrich), DC Protein Assay (Bio-Rad), Dialysed Foetal Bovine Serum (FBS; Life Technologies), Schneider’s Insect Medium (Sigma-Aldrich), Schneider’s Drosophila Medium without L-Methionine (PAN Biotech), Dulbecco Phosphate Buffered Saline (DPBS, Gibco), 1M Triethylammonium bicarbonate (TEAB) buffer (Sigma Aldrich), NeutrAvidin-Agarose beads (Thermo Scientific), iTRAQ Reagents Multiplex Kit (Sigma Aldrich), Optima LC-MS Grade Trifluoroacetic acid (TFA; Fisher Scientific), Optima LC-MS Grade Formic acid (FA; Fisher Scientific), Optima LC-MS Grade Acetonitrile (ACN; Fisher Scientific), Optima LC-MS Grade Methanol (MeOH; Fisher Scientific) and Sequencing Grade Modified Trypsin (Promega). .. Culturing of L . mexicana promastigotes Promastigote form of L . mexicana strain M379 (MNYC/BC/62/M379) were grown in T-25 flasks at 26 o C in Schneider’s Insect Medium (Sigma-Aldrich) supplemented with 0.4g/L NaHCO3 , 0.6g/L anhydrous CaCl2 and 10% FBS (pH 7.2).

    Article Title: Integrated Quantitative Proteomics and Metabolome Profiling Reveal MSMEG_6171 Overexpression Perturbing Lipid Metabolism of Mycobacterium smegmatis Leading to Increased Vancomycin Resistance
    Article Snippet: .. Proteomics AnalysisProteins were extracted, digested, and labeled using the iTRAQ(R) Reagents Multiplex Kit (sigma) following the manufacturer’s instructions. .. Chromatography–mass spectrometry (LC-MS) and bioinformatics analysis were performed according to previously published procedures with some modifications ( ).

    Expressing:

    Article Title: Integrated Datasets of Proteomic and Metabolomic Biomarkers to Predict Its Impacts on Comorbidities of Type 2 Diabetes Mellitus
    Article Snippet: .. For the protein expression profiling experiment, serum samples were delipidated according to the protocol described by Cham and Knowles in preparation for iTRAQ analysis. .. ProteoExtract Albumin/IgG (from Calbiochem), and Vivapure anti-HSA-IgG kits were used to evaluate the efficiency of high abundance protein depletion from serum samples.

    Protein Extraction:

    Article Title: Proteomic study of hypothalamus in pigs exposed to heat stress
    Article Snippet: .. Protein extraction and quantification, iTRAQ labeling, and strong Cation exchange (SCX) fractionation Frozen hypothalamus tissue from control and heat stress were exposed to liquid nitrogen and were ground into fine powder using a mortar. .. The powder (~ 100 mg per pig) was transferred to sterile tubes with 1 mL of lysis buffer (pH 8.5; containing 7 m urea, 2 m thiourea, 4% CHAPS, 40 mm Tris-HCl, 10 mm dithiothreitol) for 5 min. Then, the tissue was homogenized with an Ultrasonic Cell Disruptor (VCX130, USA) at 20% power output for 10 min with 2/4-s on/off cycles.

    Labeling:

    Article Title: Cyclosporine A inhibits MRTF-SRF signalling through Na+/K+ ATPase inhibition Actin remodelling
    Article Snippet: .. Cell lysates were incubated on ice for 30 min and centrifuged for 15 min at 21000 g. Supernatants were stored until protein concentration was measured using the Bradford colorimetric method and iTRAQ labeling. ..

    Article Title: Proteomic study of hypothalamus in pigs exposed to heat stress
    Article Snippet: .. Protein extraction and quantification, iTRAQ labeling, and strong Cation exchange (SCX) fractionation Frozen hypothalamus tissue from control and heat stress were exposed to liquid nitrogen and were ground into fine powder using a mortar. .. The powder (~ 100 mg per pig) was transferred to sterile tubes with 1 mL of lysis buffer (pH 8.5; containing 7 m urea, 2 m thiourea, 4% CHAPS, 40 mm Tris-HCl, 10 mm dithiothreitol) for 5 min. Then, the tissue was homogenized with an Ultrasonic Cell Disruptor (VCX130, USA) at 20% power output for 10 min with 2/4-s on/off cycles.

    Article Title: Proteomic analysis of calcified abdominal and thoracic aortic aneurysms.
    Article Snippet: .. Denaturation, reduction, trypsin digestion, and iTRAQ labeling. .. Labeling with iTRAQ was performed using iTRAQ™ Reagent from AB Sciex as described in the manufacturer's instructions.

    Article Title: Integrated Quantitative Proteomics and Metabolome Profiling Reveal MSMEG_6171 Overexpression Perturbing Lipid Metabolism of Mycobacterium smegmatis Leading to Increased Vancomycin Resistance
    Article Snippet: .. Proteomics AnalysisProteins were extracted, digested, and labeled using the iTRAQ(R) Reagents Multiplex Kit (sigma) following the manufacturer’s instructions. .. Chromatography–mass spectrometry (LC-MS) and bioinformatics analysis were performed according to previously published procedures with some modifications ( ).

    Sequencing:

    Article Title: A BONCAT-iTRAQ method enables temporally resolved quantitative profiling of newly synthesised proteins in Leishmania mexicana parasites during starvation
    Article Snippet: .. Chemicals and reagents L-Azidohomoalanine (AHA; Iris Biotech GmbH), Cycloheximide (CHX; ACROS Organics), Tris (2-carboxyethy)phosphine hydrochloride (TCEP; Sigma Aldrich), 5-Tetramethylrhodamine-Alkyne (TAMRA-Alkyne; Jena Bioscience), Acetylene-PEG4-Biotin (Biotin-Alkyne; Jena Bioscience), Tris [(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA; Sigma Aldrich), Dimethyl sulfoxide (DMSO; Sigma Aldrich), Copper sulphate (CuSO4 ; Sigma Aldrich), 2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris; Sigma Aldrich), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES; Sigma Aldrich), Sodium chloride (NaCl; Fisher Scientific), Sodium dodecyl sulphate (SDS; Fisher Scientific), Sodium bicarbonate (NaHCO3 ; ACROS Organics), Calcium chloride (CaCl2 ; Sigma Aldrich), Urea (Sigma Aldrich), 1,4-Dithiothreitol (DTT; Sigma Aldrich), 2-Chloroacetamide (CAA; Sigma Aldrich), L-Glutamine solution (Sigma Aldrich), Benzonase (Sigma Aldrich), DC Protein Assay (Bio-Rad), Dialysed Foetal Bovine Serum (FBS; Life Technologies), Schneider’s Insect Medium (Sigma-Aldrich), Schneider’s Drosophila Medium without L-Methionine (PAN Biotech), Dulbecco Phosphate Buffered Saline (DPBS, Gibco), 1M Triethylammonium bicarbonate (TEAB) buffer (Sigma Aldrich), NeutrAvidin-Agarose beads (Thermo Scientific), iTRAQ Reagents Multiplex Kit (Sigma Aldrich), Optima LC-MS Grade Trifluoroacetic acid (TFA; Fisher Scientific), Optima LC-MS Grade Formic acid (FA; Fisher Scientific), Optima LC-MS Grade Acetonitrile (ACN; Fisher Scientific), Optima LC-MS Grade Methanol (MeOH; Fisher Scientific) and Sequencing Grade Modified Trypsin (Promega). .. Culturing of L . mexicana promastigotes Promastigote form of L . mexicana strain M379 (MNYC/BC/62/M379) were grown in T-25 flasks at 26 o C in Schneider’s Insect Medium (Sigma-Aldrich) supplemented with 0.4g/L NaHCO3 , 0.6g/L anhydrous CaCl2 and 10% FBS (pH 7.2).

    Incubation:

    Article Title: Cyclosporine A inhibits MRTF-SRF signalling through Na+/K+ ATPase inhibition Actin remodelling
    Article Snippet: .. Cell lysates were incubated on ice for 30 min and centrifuged for 15 min at 21000 g. Supernatants were stored until protein concentration was measured using the Bradford colorimetric method and iTRAQ labeling. ..

    Article Title: Cyclosporine A inhibits MRTF-SRF signalling through Na+/K+ ATPase inhibition Actin remodelling
    Article Snippet: .. After digestion, samples were incubated with iTRAQ tags (iTRAQ Reagents Multi-plex kits, 4-plex, #4352135, Sigma-Aldrich) – one tag per drug exposure condition and five different tag/condition associations over five experiments – for 1 h at room temperature. .. Labeled samples were mixed and separated into 12 fractions by isoelectric focusing (OFFGEL 3100 Fractionator, Agilent Technologies, Santa Clara, CA) for 24 h at increasing voltage and steady intensity of 50 μA in a 3-10 pH IPG strip.

    Fractionation:

    Article Title: Proteomic study of hypothalamus in pigs exposed to heat stress
    Article Snippet: .. Protein extraction and quantification, iTRAQ labeling, and strong Cation exchange (SCX) fractionation Frozen hypothalamus tissue from control and heat stress were exposed to liquid nitrogen and were ground into fine powder using a mortar. .. The powder (~ 100 mg per pig) was transferred to sterile tubes with 1 mL of lysis buffer (pH 8.5; containing 7 m urea, 2 m thiourea, 4% CHAPS, 40 mm Tris-HCl, 10 mm dithiothreitol) for 5 min. Then, the tissue was homogenized with an Ultrasonic Cell Disruptor (VCX130, USA) at 20% power output for 10 min with 2/4-s on/off cycles.

    Liquid Chromatography with Mass Spectroscopy:

    Article Title: A BONCAT-iTRAQ method enables temporally resolved quantitative profiling of newly synthesised proteins in Leishmania mexicana parasites during starvation
    Article Snippet: .. Chemicals and reagents L-Azidohomoalanine (AHA; Iris Biotech GmbH), Cycloheximide (CHX; ACROS Organics), Tris (2-carboxyethy)phosphine hydrochloride (TCEP; Sigma Aldrich), 5-Tetramethylrhodamine-Alkyne (TAMRA-Alkyne; Jena Bioscience), Acetylene-PEG4-Biotin (Biotin-Alkyne; Jena Bioscience), Tris [(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA; Sigma Aldrich), Dimethyl sulfoxide (DMSO; Sigma Aldrich), Copper sulphate (CuSO4 ; Sigma Aldrich), 2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris; Sigma Aldrich), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES; Sigma Aldrich), Sodium chloride (NaCl; Fisher Scientific), Sodium dodecyl sulphate (SDS; Fisher Scientific), Sodium bicarbonate (NaHCO3 ; ACROS Organics), Calcium chloride (CaCl2 ; Sigma Aldrich), Urea (Sigma Aldrich), 1,4-Dithiothreitol (DTT; Sigma Aldrich), 2-Chloroacetamide (CAA; Sigma Aldrich), L-Glutamine solution (Sigma Aldrich), Benzonase (Sigma Aldrich), DC Protein Assay (Bio-Rad), Dialysed Foetal Bovine Serum (FBS; Life Technologies), Schneider’s Insect Medium (Sigma-Aldrich), Schneider’s Drosophila Medium without L-Methionine (PAN Biotech), Dulbecco Phosphate Buffered Saline (DPBS, Gibco), 1M Triethylammonium bicarbonate (TEAB) buffer (Sigma Aldrich), NeutrAvidin-Agarose beads (Thermo Scientific), iTRAQ Reagents Multiplex Kit (Sigma Aldrich), Optima LC-MS Grade Trifluoroacetic acid (TFA; Fisher Scientific), Optima LC-MS Grade Formic acid (FA; Fisher Scientific), Optima LC-MS Grade Acetonitrile (ACN; Fisher Scientific), Optima LC-MS Grade Methanol (MeOH; Fisher Scientific) and Sequencing Grade Modified Trypsin (Promega). .. Culturing of L . mexicana promastigotes Promastigote form of L . mexicana strain M379 (MNYC/BC/62/M379) were grown in T-25 flasks at 26 o C in Schneider’s Insect Medium (Sigma-Aldrich) supplemented with 0.4g/L NaHCO3 , 0.6g/L anhydrous CaCl2 and 10% FBS (pH 7.2).

    Protein Concentration:

    Article Title: Cyclosporine A inhibits MRTF-SRF signalling through Na+/K+ ATPase inhibition Actin remodelling
    Article Snippet: .. Cell lysates were incubated on ice for 30 min and centrifuged for 15 min at 21000 g. Supernatants were stored until protein concentration was measured using the Bradford colorimetric method and iTRAQ labeling. ..

    Modification:

    Article Title: A BONCAT-iTRAQ method enables temporally resolved quantitative profiling of newly synthesised proteins in Leishmania mexicana parasites during starvation
    Article Snippet: .. Chemicals and reagents L-Azidohomoalanine (AHA; Iris Biotech GmbH), Cycloheximide (CHX; ACROS Organics), Tris (2-carboxyethy)phosphine hydrochloride (TCEP; Sigma Aldrich), 5-Tetramethylrhodamine-Alkyne (TAMRA-Alkyne; Jena Bioscience), Acetylene-PEG4-Biotin (Biotin-Alkyne; Jena Bioscience), Tris [(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA; Sigma Aldrich), Dimethyl sulfoxide (DMSO; Sigma Aldrich), Copper sulphate (CuSO4 ; Sigma Aldrich), 2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris; Sigma Aldrich), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES; Sigma Aldrich), Sodium chloride (NaCl; Fisher Scientific), Sodium dodecyl sulphate (SDS; Fisher Scientific), Sodium bicarbonate (NaHCO3 ; ACROS Organics), Calcium chloride (CaCl2 ; Sigma Aldrich), Urea (Sigma Aldrich), 1,4-Dithiothreitol (DTT; Sigma Aldrich), 2-Chloroacetamide (CAA; Sigma Aldrich), L-Glutamine solution (Sigma Aldrich), Benzonase (Sigma Aldrich), DC Protein Assay (Bio-Rad), Dialysed Foetal Bovine Serum (FBS; Life Technologies), Schneider’s Insect Medium (Sigma-Aldrich), Schneider’s Drosophila Medium without L-Methionine (PAN Biotech), Dulbecco Phosphate Buffered Saline (DPBS, Gibco), 1M Triethylammonium bicarbonate (TEAB) buffer (Sigma Aldrich), NeutrAvidin-Agarose beads (Thermo Scientific), iTRAQ Reagents Multiplex Kit (Sigma Aldrich), Optima LC-MS Grade Trifluoroacetic acid (TFA; Fisher Scientific), Optima LC-MS Grade Formic acid (FA; Fisher Scientific), Optima LC-MS Grade Acetonitrile (ACN; Fisher Scientific), Optima LC-MS Grade Methanol (MeOH; Fisher Scientific) and Sequencing Grade Modified Trypsin (Promega). .. Culturing of L . mexicana promastigotes Promastigote form of L . mexicana strain M379 (MNYC/BC/62/M379) were grown in T-25 flasks at 26 o C in Schneider’s Insect Medium (Sigma-Aldrich) supplemented with 0.4g/L NaHCO3 , 0.6g/L anhydrous CaCl2 and 10% FBS (pH 7.2).

    Western Blot:

    Article Title: iTRAQ-based proteomics analysis reveals the deregulated proteins related to liver toxicity induced by melamine with or without cyanuric acid in mice.
    Article Snippet: .. The administration of melamine alone or its combination with cyanuric acid was shown to have certain liver toxicity. .. The administration of melamine alone or its combination with cyanuric acid was shown to have certain liver toxicity.

    Cellular Antioxidant Activity Assay:

    Article Title: A BONCAT-iTRAQ method enables temporally resolved quantitative profiling of newly synthesised proteins in Leishmania mexicana parasites during starvation
    Article Snippet: .. Chemicals and reagents L-Azidohomoalanine (AHA; Iris Biotech GmbH), Cycloheximide (CHX; ACROS Organics), Tris (2-carboxyethy)phosphine hydrochloride (TCEP; Sigma Aldrich), 5-Tetramethylrhodamine-Alkyne (TAMRA-Alkyne; Jena Bioscience), Acetylene-PEG4-Biotin (Biotin-Alkyne; Jena Bioscience), Tris [(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA; Sigma Aldrich), Dimethyl sulfoxide (DMSO; Sigma Aldrich), Copper sulphate (CuSO4 ; Sigma Aldrich), 2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris; Sigma Aldrich), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES; Sigma Aldrich), Sodium chloride (NaCl; Fisher Scientific), Sodium dodecyl sulphate (SDS; Fisher Scientific), Sodium bicarbonate (NaHCO3 ; ACROS Organics), Calcium chloride (CaCl2 ; Sigma Aldrich), Urea (Sigma Aldrich), 1,4-Dithiothreitol (DTT; Sigma Aldrich), 2-Chloroacetamide (CAA; Sigma Aldrich), L-Glutamine solution (Sigma Aldrich), Benzonase (Sigma Aldrich), DC Protein Assay (Bio-Rad), Dialysed Foetal Bovine Serum (FBS; Life Technologies), Schneider’s Insect Medium (Sigma-Aldrich), Schneider’s Drosophila Medium without L-Methionine (PAN Biotech), Dulbecco Phosphate Buffered Saline (DPBS, Gibco), 1M Triethylammonium bicarbonate (TEAB) buffer (Sigma Aldrich), NeutrAvidin-Agarose beads (Thermo Scientific), iTRAQ Reagents Multiplex Kit (Sigma Aldrich), Optima LC-MS Grade Trifluoroacetic acid (TFA; Fisher Scientific), Optima LC-MS Grade Formic acid (FA; Fisher Scientific), Optima LC-MS Grade Acetonitrile (ACN; Fisher Scientific), Optima LC-MS Grade Methanol (MeOH; Fisher Scientific) and Sequencing Grade Modified Trypsin (Promega). .. Culturing of L . mexicana promastigotes Promastigote form of L . mexicana strain M379 (MNYC/BC/62/M379) were grown in T-25 flasks at 26 o C in Schneider’s Insect Medium (Sigma-Aldrich) supplemented with 0.4g/L NaHCO3 , 0.6g/L anhydrous CaCl2 and 10% FBS (pH 7.2).

    DC Protein Assay:

    Article Title: A BONCAT-iTRAQ method enables temporally resolved quantitative profiling of newly synthesised proteins in Leishmania mexicana parasites during starvation
    Article Snippet: .. Chemicals and reagents L-Azidohomoalanine (AHA; Iris Biotech GmbH), Cycloheximide (CHX; ACROS Organics), Tris (2-carboxyethy)phosphine hydrochloride (TCEP; Sigma Aldrich), 5-Tetramethylrhodamine-Alkyne (TAMRA-Alkyne; Jena Bioscience), Acetylene-PEG4-Biotin (Biotin-Alkyne; Jena Bioscience), Tris [(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA; Sigma Aldrich), Dimethyl sulfoxide (DMSO; Sigma Aldrich), Copper sulphate (CuSO4 ; Sigma Aldrich), 2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris; Sigma Aldrich), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES; Sigma Aldrich), Sodium chloride (NaCl; Fisher Scientific), Sodium dodecyl sulphate (SDS; Fisher Scientific), Sodium bicarbonate (NaHCO3 ; ACROS Organics), Calcium chloride (CaCl2 ; Sigma Aldrich), Urea (Sigma Aldrich), 1,4-Dithiothreitol (DTT; Sigma Aldrich), 2-Chloroacetamide (CAA; Sigma Aldrich), L-Glutamine solution (Sigma Aldrich), Benzonase (Sigma Aldrich), DC Protein Assay (Bio-Rad), Dialysed Foetal Bovine Serum (FBS; Life Technologies), Schneider’s Insect Medium (Sigma-Aldrich), Schneider’s Drosophila Medium without L-Methionine (PAN Biotech), Dulbecco Phosphate Buffered Saline (DPBS, Gibco), 1M Triethylammonium bicarbonate (TEAB) buffer (Sigma Aldrich), NeutrAvidin-Agarose beads (Thermo Scientific), iTRAQ Reagents Multiplex Kit (Sigma Aldrich), Optima LC-MS Grade Trifluoroacetic acid (TFA; Fisher Scientific), Optima LC-MS Grade Formic acid (FA; Fisher Scientific), Optima LC-MS Grade Acetonitrile (ACN; Fisher Scientific), Optima LC-MS Grade Methanol (MeOH; Fisher Scientific) and Sequencing Grade Modified Trypsin (Promega). .. Culturing of L . mexicana promastigotes Promastigote form of L . mexicana strain M379 (MNYC/BC/62/M379) were grown in T-25 flasks at 26 o C in Schneider’s Insect Medium (Sigma-Aldrich) supplemented with 0.4g/L NaHCO3 , 0.6g/L anhydrous CaCl2 and 10% FBS (pH 7.2).