sequencing grade modified trypsin  (Promega)

 
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    Name:
    Sequencing Grade Modified Trypsin, Frozen, 100ug
    Description:

    Catalog Number:
    V5113
    Price:
    None
    Score:
    85
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    Promega sequencing grade modified trypsin

    https://www.bioz.com/result/sequencing grade modified trypsin/product/Promega
    Average 99 stars, based on 559 article reviews
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    sequencing grade modified trypsin - by Bioz Stars, 2020-01
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    Centrifugation:

    Article Title: Chemoproteomic profiling of host and pathogen enzymes active in cholera
    Article Snippet: The washed beads were suspended in 6 M urea in PBS (500 μL) and dithiothreitol (10 mM final concentration, diluted from a 20X stock in water) and incubated at 65 °C for 15 min. Iodoacetamide (20 mM final concentration, diluted from a 20X stock in water) was then reacted with the samples at 37 °C for 30 min. .. Following reduction and alkylation, the beads were pelleted by centrifugation (1400 × g, 4 °C, 3 min) and suspended in 200 μL of 2 M urea, 1 mM CaCl2 (diluted from a 100X stock in water), and sequencing-grade modified trypsin (2 μg; Promega) in PBS. .. The digested peptides were separated from the beads using a Micro Bio-Spin column (BioRad).

    Article Title: Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like
    Article Snippet: In-solution tryptic digests of the samples were prepared using an enhanced filter-aided sample preparation (eFASP) method . .. Proteins were digested in the filter unit in 100 μl digestion buffer with 200 ng sequencing-grade modified trypsin (Promega) at 37 °C for 12–16 h. Peptides were isolated by centrifugation and were extracted with ethyl acetate to remove remaining deoxycholic acid . .. Three unique peptides for each protein of interest were chosen for isolation based on previous data-dependent discovery data or from online peptide databases ( www.peptideatlas.org , www.thegpm.org ).

    Article Title:
    Article Snippet: The tubes were briefly vortexed and incubated on ice for 40 min, with brief vortexing of the sample every 5 min. After incubation, the samples were centrifuged at 13,000 rpm, and the supernatant was transferred to new tubes and subjected to a second centrifugation. .. Next, sequencing-grade modified trypsin (Promega, Madison, WI) was added at 20 μg per milligram of protein for 16 h at 37 °C.

    Article Title: The SKP1-Cullin-F-box E3 ligase βTrCP and CDK2 cooperate to control STIL abundance and centriole number
    Article Snippet: After centrifugation (15 min, 4°C, 16 000g ), cell extracts were incubated at 4°C with Anti-FLAG M2 Affinity Gel (Sigma-Aldrich) for 2 h, thereafter beads were washed three times with Tris lysis buffer, followed by three washes with phosphate-buffered saline. .. Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega) overnight at 37°C.

    BIA-KA:

    Article Title: The quantitative and condition-dependent Escherichia coli proteome
    Article Snippet: Then, the samples were diluted with 100mM ammoniumbicarbonate buffer to a final sodium deoxycholate (urea) concentration of 1% (1.6M) for dataset 2 (1) samples, respectively, and further digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, Wisconsin) over night at 37°C. .. Then, the samples were diluted with 100mM ammoniumbicarbonate buffer to a final sodium deoxycholate (urea) concentration of 1% (1.6M) for dataset 2 (1) samples, respectively, and further digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, Wisconsin) over night at 37°C.

    Article Title: Cx43-Associated Secretome and Interactome Reveal Synergistic Mechanisms for Glioma Migration and MMP3 Activation
    Article Snippet: Total protein quantity was determined by BCA (Pierce/Thermo Fisher Scientific, Rockford, IL, United States). .. Protein digestions were conducted overnight (37°C) with sequencing-grade modified trypsin (1 μg/50 μg protein; Promega, Madison, WI, United States).

    Article Title: The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan
    Article Snippet: Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, WI, USA) overnight at 37 °C. .. Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, WI, USA) overnight at 37 °C.

    Article Title: The SKP1-Cullin-F-box E3 ligase βTrCP and CDK2 cooperate to control STIL abundance and centriole number
    Article Snippet: Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega) overnight at 37°C. .. Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega) overnight at 37°C.

    Incubation:

    Article Title: The quantitative and condition-dependent Escherichia coli proteome
    Article Snippet: After quenching the reaction with 12 mM N-acetyl- cysteine, the proteins were proteolyzed for 4h at 37°C using sequencing-grade Lys-C (Wako Chemicals) at 1/200 w/w. .. Then, the samples were diluted with 100mM ammoniumbicarbonate buffer to a final sodium deoxycholate (urea) concentration of 1% (1.6M) for dataset 2 (1) samples, respectively, and further digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, Wisconsin) over night at 37°C. .. The samples were acidified with 2M HCl to a final concentration of 50mM, incubated for 15min at 37°C and the precipitated detergent removed by centrifugation at 10,000g for 15min.

    Article Title: Complex formation of APP with GABAB receptors links axonal trafficking to amyloidogenic processing
    Article Snippet: For a quantitative comparison of GBRs in two samples (Fig. ), a mixture of anti-GB antibodies including rabbit anti-GB1 (322102, Synaptic Systems), rabbit anti-GB2 (322203, Synaptic Systems), guinea pig anti-GB2 (322204/5, Synaptic Systems)) was applied to isolate the complete pool of receptor protein complexes, which was controlled by immunoblot analysis of the respective supernatant after antibody incubation. .. Lanes were cut into two sections (high and low MW) and digested with sequencing-grade modified trypsin (Promega, Mannheim, Germany).

    Article Title: Chemoproteomic profiling of host and pathogen enzymes active in cholera
    Article Snippet: Samples were adjusted to 0.5 mg/mL in PBS and divided into 2 technical replicates of equal volume (90 μL). .. Samples were incubated with sequencing-grade modified trypsin (2 μg; Promega) and 1 mM CaCl2 and digested overnight at 37 °C. .. Digested peptides were treated with formic acid (5 μL) and centrifuged (100,000 × g, 4 °C, 1 h) to remove any remaining particulates.

    Article Title: Chemoproteomic profiling of host and pathogen enzymes active in cholera
    Article Snippet: The washed beads were suspended in 6 M urea in PBS (500 μL) and dithiothreitol (10 mM final concentration, diluted from a 20X stock in water) and incubated at 65 °C for 15 min. Iodoacetamide (20 mM final concentration, diluted from a 20X stock in water) was then reacted with the samples at 37 °C for 30 min. .. Following reduction and alkylation, the beads were pelleted by centrifugation (1400 × g, 4 °C, 3 min) and suspended in 200 μL of 2 M urea, 1 mM CaCl2 (diluted from a 100X stock in water), and sequencing-grade modified trypsin (2 μg; Promega) in PBS.

    Article Title: Quantitative Fluorescent Labeling of Aldehyde-Tagged Proteins for Single-Molecule Imaging
    Article Snippet: The extinction coefficient of Ald6N -PolBI and -DinB under native condition was thus determined to be 81,300–81,900 and 30,700–31,000 M−1 cm−1 , respectively. .. Aldehyde-tagged polymerase was mixed in a 50:1 weight ratio with sequencing-grade modified trypsin (Promega) in a pH 7.8 buffer containing 100 mM ammonium bicarbonate and incubated at 37 °C overnight. .. The product was separated on a nanoLC-2D system (Eksigent) at a flow rate of 300 nl/min, using a gradient from 95% A containing 5% acetonitrile and 0.2% formic acid and 5% buffer B containing 95% acetonitrile and 0.2% formic acid, to 55% A and 45% B in 50 min, and then to 15% A and 85% B in 10 min.

    Article Title: The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan
    Article Snippet: After quenching the reaction with 12 mM N-acetyl-cysteine the samples were diluted with 100 mM ammonium bicarbonate buffer to a final DOC concentration of 1%. .. Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, WI, USA) overnight at 37 °C. .. Then, the samples were acidified with 2 M HCl to a final concentration of 50 mM, incubated for 15 min at 37 °C and the precipitated detergent removed by centrifugation at 10,000×g for 15 min.

    Article Title: Comprehensive evaluation of coding region point mutations in microsatellite‐unstable colorectal cancer
    Article Snippet: After the final wash, the beads were resuspended in 2 × 300 μl of elution buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 50 mM NaF, 5 mM EDTA, 0.5 mM biotin) and incubated for 5 min, and eluates were collected, followed by reduction in the cysteine bonds with 5 mM Tris(2‐carboxyethyl)phosphine (TCEP) for 20 min at 37°C and alkylation with 10 mM iodoacetamide (at room temperature, in the dark). .. Proteins were digested overnight at 37°C with sequencing‐grade modified trypsin (Promega, Madison, WI, USA).

    Article Title: Pathogen signatures activate a ubiquitinylation pathway that modulates function of the metabolic checkpoint kinase mTOR
    Article Snippet: Lysate supernatants from a high-speed spin (~4mls) were combined with 100µl of Streptavidin-agarose beads (Thermo scientific) pre-equilibrated in urea buffer and rotated at room temperature for 2hrs. .. Beads were washed 6X with 8M urea buffer, resuspended in 50µl of 8M urea buffer containing 5mM DTT and incubated at 60C for 30min, followed by addition of 2-chloroacetamide to 50 mM final concentration and incubation at room temperature for 60 min. On-beads trypsin digestion were performed by diluting samples to 2M urea, adding 100ng of sequencing-grade modified trypsin (Promega) and incubating for 24hrs at 37C. .. LC-MS/MS and subsequent analysis was carried out at the Yale W.M.

    Article Title: AMPA-receptor specific biogenesis complexes control synaptic transmission and intellectual ability
    Article Snippet: After 2 h of incubation and two brief washes, proteins were eluted, shortly run on SDS–PAGE gels and silver-stained. .. Lanes were cut into two sections (high and low MW) and digested with sequencing-grade modified trypsin (Promega, Mannheim, Germany).

    Article Title: Systematic verification of bladder cancer-associated tissue protein biomarker candidates in clinical urine specimens
    Article Snippet: The sample was reduced by adding 12.4 μl of 50 mM Tris(2-carboxyethyl)phosphine (TCEP; final concentration, 5 mM) and incubating at 60°C for 30 min. .. The sample was then alkylated by adding 13.75 μl of 200 mM iodoacetamide (final concentration, 10 mM) and incubating at room temperature in the dark for 30 min. Sequencing-grade modified trypsin (Promega, Madison, WI, USA) was then added at a 1:20 (enzyme/substrate) ratio, and the samples were incubated at 37°C for 9 h. Tryptic peptides were stored at −20°C for subsequent dimethyl labeling. .. Tryptic peptide samples (25 μg in 100 μl of 0.1 M triethylammonium bicarbonate) were mixed with 7.5 μl of freshly prepared sodium cyanoborohydride (2 M).

    Article Title:
    Article Snippet: The tubes were briefly vortexed and incubated on ice for 40 min, with brief vortexing of the sample every 5 min. After incubation, the samples were centrifuged at 13,000 rpm, and the supernatant was transferred to new tubes and subjected to a second centrifugation. .. Next, sequencing-grade modified trypsin (Promega, Madison, WI) was added at 20 μg per milligram of protein for 16 h at 37 °C.

    Article Title: The SKP1-Cullin-F-box E3 ligase βTrCP and CDK2 cooperate to control STIL abundance and centriole number
    Article Snippet: Sample aliquots containing 50 µg of total proteins were reduced for 10 min at 95°C and alkylated by adding chloroacetamide to 15 mM final concentration for 30 min at 37°C. .. Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega) overnight at 37°C. .. An aliquot of a heavy reference peptide mix containing 16 chemically synthesized proteotypic peptides (AQUA-grade, Thermo Fisher Scientific, see Bauer et al .

    Infection:

    Article Title: Pathogen signatures activate a ubiquitinylation pathway that modulates function of the metabolic checkpoint kinase mTOR
    Article Snippet: Total of 1×108 Raw-Ub or Raw-UbG76V were seeded in 10cm dishes (2×107 cells/plate) and infected with the Lp01 or dotA strains for 1hrs MOI of 40, then washed 3X with warm media to remove extracellular bacteria and cultured in the presence of 1nM epoxomicin (Enzo Life-sciences) for additional 3hrs. .. Beads were washed 6X with 8M urea buffer, resuspended in 50µl of 8M urea buffer containing 5mM DTT and incubated at 60C for 30min, followed by addition of 2-chloroacetamide to 50 mM final concentration and incubation at room temperature for 60 min. On-beads trypsin digestion were performed by diluting samples to 2M urea, adding 100ng of sequencing-grade modified trypsin (Promega) and incubating for 24hrs at 37C.

    Mass Spectrometry:

    Article Title: Dectin-1 Activation on Macrophages by Galectin-9 Promotes Pancreatic Carcinoma and Peritumoral Immune-Tolerance
    Article Snippet: Paragraph title: Enrichment of endogenous Dectin-1 ligands and mass spectroscopy ... Sequencing grade-modified trypsin (300ng; Promega, Madison, WI) was added to the dried gel pieces.

    Article Title: Complex formation of APP with GABAB receptors links axonal trafficking to amyloidogenic processing
    Article Snippet: After two washes, proteins were eluted and the majority processed for MS-analysis. .. Lanes were cut into two sections (high and low MW) and digested with sequencing-grade modified trypsin (Promega, Mannheim, Germany).

    Article Title: Chemoproteomic profiling of host and pathogen enzymes active in cholera
    Article Snippet: Samples were incubated with sequencing-grade modified trypsin (2 μg; Promega) and 1 mM CaCl2 and digested overnight at 37 °C. .. Samples were incubated with sequencing-grade modified trypsin (2 μg; Promega) and 1 mM CaCl2 and digested overnight at 37 °C.

    Article Title: Chemoproteomic profiling of host and pathogen enzymes active in cholera
    Article Snippet: Following reduction and alkylation, the beads were pelleted by centrifugation (1400 × g, 4 °C, 3 min) and suspended in 200 μL of 2 M urea, 1 mM CaCl2 (diluted from a 100X stock in water), and sequencing-grade modified trypsin (2 μg; Promega) in PBS. .. Following reduction and alkylation, the beads were pelleted by centrifugation (1400 × g, 4 °C, 3 min) and suspended in 200 μL of 2 M urea, 1 mM CaCl2 (diluted from a 100X stock in water), and sequencing-grade modified trypsin (2 μg; Promega) in PBS.

    Article Title: Type VI secretion system sheath inter‐subunit interactions modulate its contraction
    Article Snippet: Paragraph title: Mass spectrometry ... Proteins were digested using sequencing‐grade modified trypsin (1/250, w/w; Promega, USA) overnight at 37°C.

    Article Title: Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like
    Article Snippet: Paragraph title: Targeted mass spectrometry of purified hair bundles ... Proteins were digested in the filter unit in 100 μl digestion buffer with 200 ng sequencing-grade modified trypsin (Promega) at 37 °C for 12–16 h. Peptides were isolated by centrifugation and were extracted with ethyl acetate to remove remaining deoxycholic acid .

    Article Title: Mitochondrial PKM2 regulates oxidative stress-induced apoptosis by stabilizing Bcl2
    Article Snippet: Paragraph title: Mass spectrometry analysis ... The sample was heated to 95 °C for 10 min and then allowed to cool; 100 ng of sequencing-grade modified trypsin (Promega, Madison, WI, USA) was added.

    Article Title: Quantitative Fluorescent Labeling of Aldehyde-Tagged Proteins for Single-Molecule Imaging
    Article Snippet: Paragraph title: High-Resolution Mass Spectrometry ... Aldehyde-tagged polymerase was mixed in a 50:1 weight ratio with sequencing-grade modified trypsin (Promega) in a pH 7.8 buffer containing 100 mM ammonium bicarbonate and incubated at 37 °C overnight.

    Bradford Assay:

    Article Title: Chemoproteomic profiling of host and pathogen enzymes active in cholera
    Article Snippet: PBS (120 μL) was added to each sample, and protein concentrations were measured using the Pierce Coomassie Plus (Bradford) Assay Kit (Life Technologies). .. Samples were incubated with sequencing-grade modified trypsin (2 μg; Promega) and 1 mM CaCl2 and digested overnight at 37 °C.

    Modification:

    Article Title: The quantitative and condition-dependent Escherichia coli proteome
    Article Snippet: After quenching the reaction with 12 mM N-acetyl- cysteine, the proteins were proteolyzed for 4h at 37°C using sequencing-grade Lys-C (Wako Chemicals) at 1/200 w/w. .. Then, the samples were diluted with 100mM ammoniumbicarbonate buffer to a final sodium deoxycholate (urea) concentration of 1% (1.6M) for dataset 2 (1) samples, respectively, and further digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, Wisconsin) over night at 37°C. .. The samples were acidified with 2M HCl to a final concentration of 50mM, incubated for 15min at 37°C and the precipitated detergent removed by centrifugation at 10,000g for 15min.

    Article Title: Complex formation of APP with GABAB receptors links axonal trafficking to amyloidogenic processing
    Article Snippet: Proteins were separated on SDS-PAGE gels and silver-stained. .. Lanes were cut into two sections (high and low MW) and digested with sequencing-grade modified trypsin (Promega, Mannheim, Germany). .. Influence of GBR activity on complex formation was analyzed by incubating unsolubilized membranes with 1 mM GABA or 4 µM CGP54626 in PBS buffer for 1 h at room temperature.

    Article Title: Dementia-linked amyloidosis is associated with brain protein deamidation as revealed by proteomic profiling of human brain tissues
    Article Snippet: Together, these data reveal for the first time that deamidation of S100A9 may represent a key mechanism that triggers protein aggregation and amyloid formation in the human brain. .. Sequencing-grade modified trypsin (V5111) was from Promega (Madison, WI, USA). .. Protease inhibitor cocktail tablets (cOmplete) were from Roche (Basel, Switzerland).

    Article Title: Chemoproteomic profiling of host and pathogen enzymes active in cholera
    Article Snippet: Samples were adjusted to 0.5 mg/mL in PBS and divided into 2 technical replicates of equal volume (90 μL). .. Samples were incubated with sequencing-grade modified trypsin (2 μg; Promega) and 1 mM CaCl2 and digested overnight at 37 °C. .. Digested peptides were treated with formic acid (5 μL) and centrifuged (100,000 × g, 4 °C, 1 h) to remove any remaining particulates.

    Article Title: Chemoproteomic profiling of host and pathogen enzymes active in cholera
    Article Snippet: The washed beads were suspended in 6 M urea in PBS (500 μL) and dithiothreitol (10 mM final concentration, diluted from a 20X stock in water) and incubated at 65 °C for 15 min. Iodoacetamide (20 mM final concentration, diluted from a 20X stock in water) was then reacted with the samples at 37 °C for 30 min. .. Following reduction and alkylation, the beads were pelleted by centrifugation (1400 × g, 4 °C, 3 min) and suspended in 200 μL of 2 M urea, 1 mM CaCl2 (diluted from a 100X stock in water), and sequencing-grade modified trypsin (2 μg; Promega) in PBS. .. The digested peptides were separated from the beads using a Micro Bio-Spin column (BioRad).

    Article Title: Type VI secretion system sheath inter‐subunit interactions modulate its contraction
    Article Snippet: Sheath samples (30–100 μg) were reduced with 5 mM Tris(2‐chloroethyl)phosphate, shaking for 1 h at 37°C and alkylated with 10 mM iodoacetamide, shaking for 30 min at 25°C in the dark. .. Proteins were digested using sequencing‐grade modified trypsin (1/250, w/w; Promega, USA) overnight at 37°C. .. After digestion, the samples were supplemented with trifluoroacetic acid to a final concentration of 1%.

    Article Title: Cx43-Associated Secretome and Interactome Reveal Synergistic Mechanisms for Glioma Migration and MMP3 Activation
    Article Snippet: Proteins (1 mg) were then reduced with DTT (1 μg/50 μg of proteins; 30 min at 37°C) and alkylated with iodoacetamide (IAA; 5 μg/50 μg of proteins, 20 min at 37°C). .. Protein digestions were conducted overnight (37°C) with sequencing-grade modified trypsin (1 μg/50 μg protein; Promega, Madison, WI, United States). .. Peptides were enriched by C18 solid-phase extraction (International Sorbent Technology Ltd., United Kingdom) and dried.

    Article Title: Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like
    Article Snippet: In-solution tryptic digests of the samples were prepared using an enhanced filter-aided sample preparation (eFASP) method . .. Proteins were digested in the filter unit in 100 μl digestion buffer with 200 ng sequencing-grade modified trypsin (Promega) at 37 °C for 12–16 h. Peptides were isolated by centrifugation and were extracted with ethyl acetate to remove remaining deoxycholic acid . .. Three unique peptides for each protein of interest were chosen for isolation based on previous data-dependent discovery data or from online peptide databases ( www.peptideatlas.org , www.thegpm.org ).

    Article Title: Mitochondrial PKM2 regulates oxidative stress-induced apoptosis by stabilizing Bcl2
    Article Snippet: An in vitro PKM2-phosphorylated recombinant Bcl2 was exhaustively acetylated with acetic anhydride and triethylamine in acetonitrile, evaporated to dryness, then resuspended in 50 mM ammonium bicarbonate buffer containing Rapigest (Waters Corp, MA, USA). .. The sample was heated to 95 °C for 10 min and then allowed to cool; 100 ng of sequencing-grade modified trypsin (Promega, Madison, WI, USA) was added. .. The digestion proceeded overnight at 37 °C and was analyzed by LC-MS/MS on an Obitrap-XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).

    Article Title: A Novel Prebiotic Blend Product Prevents Irritable Bowel Syndrome in Mice by Improving Gut Microbiota and Modulating Immune Response
    Article Snippet: For each sample, proteins were precipitated with ice-cold acetone, and then were re-dissolved in 100 μL TEAB (tetraethyl-ammonium bromide). .. Then proteins were tryptic digested with 1:50 sequence-grade modified trypsin (Promega, Madison, WI, USA), and the resultant peptide mixture was labeled with iTRAQ (isobaric tags for relative and absolute quantitation). .. The peptide mixture was separated at high pH, and twelve fractions were collected and each fraction dried in a vacuum concentrator for the next step.

    Article Title: The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan
    Article Snippet: After quenching the reaction with 12 mM N-acetyl-cysteine the samples were diluted with 100 mM ammonium bicarbonate buffer to a final DOC concentration of 1%. .. Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, WI, USA) overnight at 37 °C. .. Then, the samples were acidified with 2 M HCl to a final concentration of 50 mM, incubated for 15 min at 37 °C and the precipitated detergent removed by centrifugation at 10,000×g for 15 min.

    Article Title: Comprehensive evaluation of coding region point mutations in microsatellite‐unstable colorectal cancer
    Article Snippet: After the final wash, the beads were resuspended in 2 × 300 μl of elution buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 50 mM NaF, 5 mM EDTA, 0.5 mM biotin) and incubated for 5 min, and eluates were collected, followed by reduction in the cysteine bonds with 5 mM Tris(2‐carboxyethyl)phosphine (TCEP) for 20 min at 37°C and alkylation with 10 mM iodoacetamide (at room temperature, in the dark). .. Proteins were digested overnight at 37°C with sequencing‐grade modified trypsin (Promega, Madison, WI, USA). .. After quenching with 10% TFA, the samples were desalted with C18 reverse‐phase spin columns according to the manufacturer's protocol (Harvard Apparatus, Cambridge, MA, USA).

    Article Title: Pathogen signatures activate a ubiquitinylation pathway that modulates function of the metabolic checkpoint kinase mTOR
    Article Snippet: Lysate supernatants from a high-speed spin (~4mls) were combined with 100µl of Streptavidin-agarose beads (Thermo scientific) pre-equilibrated in urea buffer and rotated at room temperature for 2hrs. .. Beads were washed 6X with 8M urea buffer, resuspended in 50µl of 8M urea buffer containing 5mM DTT and incubated at 60C for 30min, followed by addition of 2-chloroacetamide to 50 mM final concentration and incubation at room temperature for 60 min. On-beads trypsin digestion were performed by diluting samples to 2M urea, adding 100ng of sequencing-grade modified trypsin (Promega) and incubating for 24hrs at 37C. .. LC-MS/MS and subsequent analysis was carried out at the Yale W.M.

    Article Title: AMPA-receptor specific biogenesis complexes control synaptic transmission and intellectual ability
    Article Snippet: After 2 h of incubation and two brief washes, proteins were eluted, shortly run on SDS–PAGE gels and silver-stained. .. Lanes were cut into two sections (high and low MW) and digested with sequencing-grade modified trypsin (Promega, Mannheim, Germany). .. The following ABs were used: anti-GluA1 (Millipore, #AB1504, 1:1,000), anti-GluA2 (NeuroMab, #75-002, 1:1,000), anti-GluA2/3 (Millipore, #07-598, 1:1,000), anti-GluA3 (Synaptic Systems, #182203, 1:1,000), anti-GluA4 (Millipore, #AB1508, 1:1,000), rabbit anti-FRRS1l-a, anti-FRRS1l-b (epitope: rat FRRS1l aa 47-66, AB generation by AbFrontiers, South Korea, 1:1,000, 15 μg), rabbit anti-FRRS1l-c, anti-FRRS1l-d (rat FRRS1l aa 27–44, generated by AbFrontiers, 1:1,000, 15 μg), anti-CPT1c-a (Santa Cruz Biotechnology, sc-139479, 1:1,000, 15 μg), anti-CPT1c-b , anti-TARP-a (anti-TARP-γ2,3; Millipore, #07-577, 15 μg), anti-TARP-b (anti-TARP-γ 2,4,8; NeuroMab, #75-252, 15 μg), anti-TARP-c (anti-TARP-γ8; Frontier Institute, #TARP γ8-Rb-Af1000, 15 μg), and IgG rabbit (Millipore, #12-370, 15 μg).

    Article Title: Systematic verification of bladder cancer-associated tissue protein biomarker candidates in clinical urine specimens
    Article Snippet: The sample was reduced by adding 12.4 μl of 50 mM Tris(2-carboxyethyl)phosphine (TCEP; final concentration, 5 mM) and incubating at 60°C for 30 min. .. The sample was then alkylated by adding 13.75 μl of 200 mM iodoacetamide (final concentration, 10 mM) and incubating at room temperature in the dark for 30 min. Sequencing-grade modified trypsin (Promega, Madison, WI, USA) was then added at a 1:20 (enzyme/substrate) ratio, and the samples were incubated at 37°C for 9 h. Tryptic peptides were stored at −20°C for subsequent dimethyl labeling. .. Tryptic peptide samples (25 μg in 100 μl of 0.1 M triethylammonium bicarbonate) were mixed with 7.5 μl of freshly prepared sodium cyanoborohydride (2 M).

    Article Title:
    Article Snippet: The samples were cooled down to room temperature before iodoacetamide was added to 10 mm , and then the samples were alkylated for 30 min at room temperature. .. Next, sequencing-grade modified trypsin (Promega, Madison, WI) was added at 20 μg per milligram of protein for 16 h at 37 °C. .. In order to ensure complete digestion, a second aliquot of trypsin (10 μg per milligram of protein) was added and the tubes were incubated for an additional 3 h at 37 °C.

    Article Title: The SKP1-Cullin-F-box E3 ligase βTrCP and CDK2 cooperate to control STIL abundance and centriole number
    Article Snippet: Sample aliquots containing 50 µg of total proteins were reduced for 10 min at 95°C and alkylated by adding chloroacetamide to 15 mM final concentration for 30 min at 37°C. .. Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega) overnight at 37°C. .. An aliquot of a heavy reference peptide mix containing 16 chemically synthesized proteotypic peptides (AQUA-grade, Thermo Fisher Scientific, see Bauer et al .

    High Performance Liquid Chromatography:

    Article Title: Type VI secretion system sheath inter‐subunit interactions modulate its contraction
    Article Snippet: Proteins were digested using sequencing‐grade modified trypsin (1/250, w/w; Promega, USA) overnight at 37°C. .. After digestion, the samples were supplemented with trifluoroacetic acid to a final concentration of 1%.

    Flow Cytometry:

    Article Title: Type VI secretion system sheath inter‐subunit interactions modulate its contraction
    Article Snippet: Proteins were digested using sequencing‐grade modified trypsin (1/250, w/w; Promega, USA) overnight at 37°C. .. 1 μg of peptides of each sample was subjected to LC‐MS analysis using a dual‐pressure LTQ‐Orbitrap Elite mass spectrometer connected to an electrospray ion source (both Thermo Fisher Scientific, Waltham, Massachusetts, USA) as described recently with a few modifications.

    Chromatography:

    Article Title: A Novel Prebiotic Blend Product Prevents Irritable Bowel Syndrome in Mice by Improving Gut Microbiota and Modulating Immune Response
    Article Snippet: Then proteins were tryptic digested with 1:50 sequence-grade modified trypsin (Promega, Madison, WI, USA), and the resultant peptide mixture was labeled with iTRAQ (isobaric tags for relative and absolute quantitation). .. Then proteins were tryptic digested with 1:50 sequence-grade modified trypsin (Promega, Madison, WI, USA), and the resultant peptide mixture was labeled with iTRAQ (isobaric tags for relative and absolute quantitation).

    Immunoprecipitation:

    Article Title: Complex formation of APP with GABAB receptors links axonal trafficking to amyloidogenic processing
    Article Snippet: 10–15 µg of the following antibodies were used for an immunoprecipitation out of 1 mg of solubilised membrane proteins: anti-APP, Ab#1, rabbit anti-APP (A8717, Sigma), Ab#2, rabbit anti-APP (ABIN1741750, Antikörper-online), Ab#3, goat anti-APP (sc-7498, Santa Cruz); anti-AJAP-1, Ab#1, sheep anti-AJAP-1 (AF7970, R & D Systems), Ab#2, rabbit anti-AJAP-1 (HPA012157, Sigma), Ab#3, goat anti-AJAP-1 (sc-163371, Santa Cruz); anti-PIANP, Ab#1, rabbit anti-PIANP (PAB21925, Abnova), Ab#2, rabbit anti-PIANP (raised against epitope: mouse PIANP aa 221–237, generated by Young in Frontier, South Korea) (Fig. ). .. Lanes were cut into two sections (high and low MW) and digested with sequencing-grade modified trypsin (Promega, Mannheim, Germany).

    Protease Inhibitor:

    Article Title: Cx43-Associated Secretome and Interactome Reveal Synergistic Mechanisms for Glioma Migration and MMP3 Activation
    Article Snippet: Protein digestions were conducted overnight (37°C) with sequencing-grade modified trypsin (1 μg/50 μg protein; Promega, Madison, WI, United States). .. Protein digestions were conducted overnight (37°C) with sequencing-grade modified trypsin (1 μg/50 μg protein; Promega, Madison, WI, United States).

    Cell Culture:

    Article Title: Pathogen signatures activate a ubiquitinylation pathway that modulates function of the metabolic checkpoint kinase mTOR
    Article Snippet: Total of 1×108 Raw-Ub or Raw-UbG76V were seeded in 10cm dishes (2×107 cells/plate) and infected with the Lp01 or dotA strains for 1hrs MOI of 40, then washed 3X with warm media to remove extracellular bacteria and cultured in the presence of 1nM epoxomicin (Enzo Life-sciences) for additional 3hrs. .. Beads were washed 6X with 8M urea buffer, resuspended in 50µl of 8M urea buffer containing 5mM DTT and incubated at 60C for 30min, followed by addition of 2-chloroacetamide to 50 mM final concentration and incubation at room temperature for 60 min. On-beads trypsin digestion were performed by diluting samples to 2M urea, adding 100ng of sequencing-grade modified trypsin (Promega) and incubating for 24hrs at 37C.

    Generated:

    Article Title: Complex formation of APP with GABAB receptors links axonal trafficking to amyloidogenic processing
    Article Snippet: 10–15 µg of the following antibodies were used for an immunoprecipitation out of 1 mg of solubilised membrane proteins: anti-APP, Ab#1, rabbit anti-APP (A8717, Sigma), Ab#2, rabbit anti-APP (ABIN1741750, Antikörper-online), Ab#3, goat anti-APP (sc-7498, Santa Cruz); anti-AJAP-1, Ab#1, sheep anti-AJAP-1 (AF7970, R & D Systems), Ab#2, rabbit anti-AJAP-1 (HPA012157, Sigma), Ab#3, goat anti-AJAP-1 (sc-163371, Santa Cruz); anti-PIANP, Ab#1, rabbit anti-PIANP (PAB21925, Abnova), Ab#2, rabbit anti-PIANP (raised against epitope: mouse PIANP aa 221–237, generated by Young in Frontier, South Korea) (Fig. ). .. Lanes were cut into two sections (high and low MW) and digested with sequencing-grade modified trypsin (Promega, Mannheim, Germany).

    Protein Concentration:

    Article Title: The quantitative and condition-dependent Escherichia coli proteome
    Article Snippet: Then, the samples were diluted with 100mM ammoniumbicarbonate buffer to a final sodium deoxycholate (urea) concentration of 1% (1.6M) for dataset 2 (1) samples, respectively, and further digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, Wisconsin) over night at 37°C. .. Then, the samples were diluted with 100mM ammoniumbicarbonate buffer to a final sodium deoxycholate (urea) concentration of 1% (1.6M) for dataset 2 (1) samples, respectively, and further digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, Wisconsin) over night at 37°C.

    Article Title: The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan
    Article Snippet: Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, WI, USA) overnight at 37 °C. .. Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, WI, USA) overnight at 37 °C.

    Article Title:
    Article Snippet: The buffer was exchanged for 0.15 m HEPES, 0.5 m urea (pH 8.0) using Zeba desalting spin columns (Pierce, Rockford, IL) before the protein concentration was determined using a Total Protein Kit, Micro Lowry (Sigma, St. Louis, MO). .. Next, sequencing-grade modified trypsin (Promega, Madison, WI) was added at 20 μg per milligram of protein for 16 h at 37 °C.

    Article Title: The SKP1-Cullin-F-box E3 ligase βTrCP and CDK2 cooperate to control STIL abundance and centriole number
    Article Snippet: Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega) overnight at 37°C. .. Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega) overnight at 37°C.

    Sequencing:

    Article Title: The quantitative and condition-dependent Escherichia coli proteome
    Article Snippet: After quenching the reaction with 12 mM N-acetyl- cysteine, the proteins were proteolyzed for 4h at 37°C using sequencing-grade Lys-C (Wako Chemicals) at 1/200 w/w. .. Then, the samples were diluted with 100mM ammoniumbicarbonate buffer to a final sodium deoxycholate (urea) concentration of 1% (1.6M) for dataset 2 (1) samples, respectively, and further digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, Wisconsin) over night at 37°C. .. The samples were acidified with 2M HCl to a final concentration of 50mM, incubated for 15min at 37°C and the precipitated detergent removed by centrifugation at 10,000g for 15min.

    Article Title: Dectin-1 Activation on Macrophages by Galectin-9 Promotes Pancreatic Carcinoma and Peritumoral Immune-Tolerance
    Article Snippet: Gel pieces were partially dehydrated with an acetonitrile rinse and further dried in a SpeedVac concentrator for 20 minutes. .. Sequencing grade-modified trypsin (300ng; Promega, Madison, WI) was added to the dried gel pieces. .. After the trypsin was absorbed, 200μl of 100mM ammonium bicarbonate was added to cover the gel pieces and digestion proceeded overnight on a shaker at 37°C.

    Article Title: Complex formation of APP with GABAB receptors links axonal trafficking to amyloidogenic processing
    Article Snippet: Proteins were separated on SDS-PAGE gels and silver-stained. .. Lanes were cut into two sections (high and low MW) and digested with sequencing-grade modified trypsin (Promega, Mannheim, Germany). .. Influence of GBR activity on complex formation was analyzed by incubating unsolubilized membranes with 1 mM GABA or 4 µM CGP54626 in PBS buffer for 1 h at room temperature.

    Article Title: Dementia-linked amyloidosis is associated with brain protein deamidation as revealed by proteomic profiling of human brain tissues
    Article Snippet: Together, these data reveal for the first time that deamidation of S100A9 may represent a key mechanism that triggers protein aggregation and amyloid formation in the human brain. .. Sequencing-grade modified trypsin (V5111) was from Promega (Madison, WI, USA). .. Protease inhibitor cocktail tablets (cOmplete) were from Roche (Basel, Switzerland).

    Article Title: Chemoproteomic profiling of host and pathogen enzymes active in cholera
    Article Snippet: Samples were adjusted to 0.5 mg/mL in PBS and divided into 2 technical replicates of equal volume (90 μL). .. Samples were incubated with sequencing-grade modified trypsin (2 μg; Promega) and 1 mM CaCl2 and digested overnight at 37 °C. .. Digested peptides were treated with formic acid (5 μL) and centrifuged (100,000 × g, 4 °C, 1 h) to remove any remaining particulates.

    Article Title: Chemoproteomic profiling of host and pathogen enzymes active in cholera
    Article Snippet: The washed beads were suspended in 6 M urea in PBS (500 μL) and dithiothreitol (10 mM final concentration, diluted from a 20X stock in water) and incubated at 65 °C for 15 min. Iodoacetamide (20 mM final concentration, diluted from a 20X stock in water) was then reacted with the samples at 37 °C for 30 min. .. Following reduction and alkylation, the beads were pelleted by centrifugation (1400 × g, 4 °C, 3 min) and suspended in 200 μL of 2 M urea, 1 mM CaCl2 (diluted from a 100X stock in water), and sequencing-grade modified trypsin (2 μg; Promega) in PBS. .. The digested peptides were separated from the beads using a Micro Bio-Spin column (BioRad).

    Article Title: Type VI secretion system sheath inter‐subunit interactions modulate its contraction
    Article Snippet: Sheath samples (30–100 μg) were reduced with 5 mM Tris(2‐chloroethyl)phosphate, shaking for 1 h at 37°C and alkylated with 10 mM iodoacetamide, shaking for 30 min at 25°C in the dark. .. Proteins were digested using sequencing‐grade modified trypsin (1/250, w/w; Promega, USA) overnight at 37°C. .. After digestion, the samples were supplemented with trifluoroacetic acid to a final concentration of 1%.

    Article Title: Cx43-Associated Secretome and Interactome Reveal Synergistic Mechanisms for Glioma Migration and MMP3 Activation
    Article Snippet: Proteins (1 mg) were then reduced with DTT (1 μg/50 μg of proteins; 30 min at 37°C) and alkylated with iodoacetamide (IAA; 5 μg/50 μg of proteins, 20 min at 37°C). .. Protein digestions were conducted overnight (37°C) with sequencing-grade modified trypsin (1 μg/50 μg protein; Promega, Madison, WI, United States). .. Peptides were enriched by C18 solid-phase extraction (International Sorbent Technology Ltd., United Kingdom) and dried.

    Article Title: Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like
    Article Snippet: In-solution tryptic digests of the samples were prepared using an enhanced filter-aided sample preparation (eFASP) method . .. Proteins were digested in the filter unit in 100 μl digestion buffer with 200 ng sequencing-grade modified trypsin (Promega) at 37 °C for 12–16 h. Peptides were isolated by centrifugation and were extracted with ethyl acetate to remove remaining deoxycholic acid . .. Three unique peptides for each protein of interest were chosen for isolation based on previous data-dependent discovery data or from online peptide databases ( www.peptideatlas.org , www.thegpm.org ).

    Article Title: Mitochondrial PKM2 regulates oxidative stress-induced apoptosis by stabilizing Bcl2
    Article Snippet: An in vitro PKM2-phosphorylated recombinant Bcl2 was exhaustively acetylated with acetic anhydride and triethylamine in acetonitrile, evaporated to dryness, then resuspended in 50 mM ammonium bicarbonate buffer containing Rapigest (Waters Corp, MA, USA). .. The sample was heated to 95 °C for 10 min and then allowed to cool; 100 ng of sequencing-grade modified trypsin (Promega, Madison, WI, USA) was added. .. The digestion proceeded overnight at 37 °C and was analyzed by LC-MS/MS on an Obitrap-XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).

    Article Title: A Novel Prebiotic Blend Product Prevents Irritable Bowel Syndrome in Mice by Improving Gut Microbiota and Modulating Immune Response
    Article Snippet: For each sample, proteins were precipitated with ice-cold acetone, and then were re-dissolved in 100 μL TEAB (tetraethyl-ammonium bromide). .. Then proteins were tryptic digested with 1:50 sequence-grade modified trypsin (Promega, Madison, WI, USA), and the resultant peptide mixture was labeled with iTRAQ (isobaric tags for relative and absolute quantitation). .. The peptide mixture was separated at high pH, and twelve fractions were collected and each fraction dried in a vacuum concentrator for the next step.

    Article Title: The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan
    Article Snippet: After quenching the reaction with 12 mM N-acetyl-cysteine the samples were diluted with 100 mM ammonium bicarbonate buffer to a final DOC concentration of 1%. .. Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, WI, USA) overnight at 37 °C. .. Then, the samples were acidified with 2 M HCl to a final concentration of 50 mM, incubated for 15 min at 37 °C and the precipitated detergent removed by centrifugation at 10,000×g for 15 min.

    Article Title: Comprehensive evaluation of coding region point mutations in microsatellite‐unstable colorectal cancer
    Article Snippet: After the final wash, the beads were resuspended in 2 × 300 μl of elution buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 50 mM NaF, 5 mM EDTA, 0.5 mM biotin) and incubated for 5 min, and eluates were collected, followed by reduction in the cysteine bonds with 5 mM Tris(2‐carboxyethyl)phosphine (TCEP) for 20 min at 37°C and alkylation with 10 mM iodoacetamide (at room temperature, in the dark). .. Proteins were digested overnight at 37°C with sequencing‐grade modified trypsin (Promega, Madison, WI, USA). .. After quenching with 10% TFA, the samples were desalted with C18 reverse‐phase spin columns according to the manufacturer's protocol (Harvard Apparatus, Cambridge, MA, USA).

    Article Title: Pathogen signatures activate a ubiquitinylation pathway that modulates function of the metabolic checkpoint kinase mTOR
    Article Snippet: Lysate supernatants from a high-speed spin (~4mls) were combined with 100µl of Streptavidin-agarose beads (Thermo scientific) pre-equilibrated in urea buffer and rotated at room temperature for 2hrs. .. Beads were washed 6X with 8M urea buffer, resuspended in 50µl of 8M urea buffer containing 5mM DTT and incubated at 60C for 30min, followed by addition of 2-chloroacetamide to 50 mM final concentration and incubation at room temperature for 60 min. On-beads trypsin digestion were performed by diluting samples to 2M urea, adding 100ng of sequencing-grade modified trypsin (Promega) and incubating for 24hrs at 37C. .. LC-MS/MS and subsequent analysis was carried out at the Yale W.M.

    Article Title: AMPA-receptor specific biogenesis complexes control synaptic transmission and intellectual ability
    Article Snippet: After 2 h of incubation and two brief washes, proteins were eluted, shortly run on SDS–PAGE gels and silver-stained. .. Lanes were cut into two sections (high and low MW) and digested with sequencing-grade modified trypsin (Promega, Mannheim, Germany). .. The following ABs were used: anti-GluA1 (Millipore, #AB1504, 1:1,000), anti-GluA2 (NeuroMab, #75-002, 1:1,000), anti-GluA2/3 (Millipore, #07-598, 1:1,000), anti-GluA3 (Synaptic Systems, #182203, 1:1,000), anti-GluA4 (Millipore, #AB1508, 1:1,000), rabbit anti-FRRS1l-a, anti-FRRS1l-b (epitope: rat FRRS1l aa 47-66, AB generation by AbFrontiers, South Korea, 1:1,000, 15 μg), rabbit anti-FRRS1l-c, anti-FRRS1l-d (rat FRRS1l aa 27–44, generated by AbFrontiers, 1:1,000, 15 μg), anti-CPT1c-a (Santa Cruz Biotechnology, sc-139479, 1:1,000, 15 μg), anti-CPT1c-b , anti-TARP-a (anti-TARP-γ2,3; Millipore, #07-577, 15 μg), anti-TARP-b (anti-TARP-γ 2,4,8; NeuroMab, #75-252, 15 μg), anti-TARP-c (anti-TARP-γ8; Frontier Institute, #TARP γ8-Rb-Af1000, 15 μg), and IgG rabbit (Millipore, #12-370, 15 μg).

    Article Title: Systematic verification of bladder cancer-associated tissue protein biomarker candidates in clinical urine specimens
    Article Snippet: The sample was reduced by adding 12.4 μl of 50 mM Tris(2-carboxyethyl)phosphine (TCEP; final concentration, 5 mM) and incubating at 60°C for 30 min. .. The sample was then alkylated by adding 13.75 μl of 200 mM iodoacetamide (final concentration, 10 mM) and incubating at room temperature in the dark for 30 min. Sequencing-grade modified trypsin (Promega, Madison, WI, USA) was then added at a 1:20 (enzyme/substrate) ratio, and the samples were incubated at 37°C for 9 h. Tryptic peptides were stored at −20°C for subsequent dimethyl labeling. .. Tryptic peptide samples (25 μg in 100 μl of 0.1 M triethylammonium bicarbonate) were mixed with 7.5 μl of freshly prepared sodium cyanoborohydride (2 M).

    Article Title:
    Article Snippet: The samples were cooled down to room temperature before iodoacetamide was added to 10 mm , and then the samples were alkylated for 30 min at room temperature. .. Next, sequencing-grade modified trypsin (Promega, Madison, WI) was added at 20 μg per milligram of protein for 16 h at 37 °C. .. In order to ensure complete digestion, a second aliquot of trypsin (10 μg per milligram of protein) was added and the tubes were incubated for an additional 3 h at 37 °C.

    Article Title: The SKP1-Cullin-F-box E3 ligase βTrCP and CDK2 cooperate to control STIL abundance and centriole number
    Article Snippet: Sample aliquots containing 50 µg of total proteins were reduced for 10 min at 95°C and alkylated by adding chloroacetamide to 15 mM final concentration for 30 min at 37°C. .. Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega) overnight at 37°C. .. An aliquot of a heavy reference peptide mix containing 16 chemically synthesized proteotypic peptides (AQUA-grade, Thermo Fisher Scientific, see Bauer et al .

    Sonication:

    Article Title: The quantitative and condition-dependent Escherichia coli proteome
    Article Snippet: The cells were disrupted by strong vortexing for 3 x 30 seconds followed by indirect sonication (100% amplitude, 0.5 cycle, 3 × 10 s) in a VialTweeter (Hielscher). .. Then, the samples were diluted with 100mM ammoniumbicarbonate buffer to a final sodium deoxycholate (urea) concentration of 1% (1.6M) for dataset 2 (1) samples, respectively, and further digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, Wisconsin) over night at 37°C.

    Article Title: Chemoproteomic profiling of host and pathogen enzymes active in cholera
    Article Snippet: Acetone-washed pellets were air-dried and resuspended by sonication in 100 μL PBS containing 2.4 M urea and 70 mM ammonium bicarbonate, treated with 15 mM dithiothreitol, and incubated at 65 °C for 15 min. Iodoacetamide (12.5 mM final concentration) was then reacted with the samples for 30 min at rt. .. Samples were incubated with sequencing-grade modified trypsin (2 μg; Promega) and 1 mM CaCl2 and digested overnight at 37 °C.

    Article Title: The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan
    Article Snippet: For each sample, 108 yeast cells were resuspended in 100 µl lysis buffer (2% sodium deoxycholate, 100 mM ammonium bicarbonate), sonicated for 2 × 10 s using a vial tweeter and spinned down. .. Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, WI, USA) overnight at 37 °C.

    Article Title: Comprehensive evaluation of coding region point mutations in microsatellite‐unstable colorectal cancer
    Article Snippet: The BioID lysates were treated with benzonase, after which they were sonicated. .. Proteins were digested overnight at 37°C with sequencing‐grade modified trypsin (Promega, Madison, WI, USA).

    Injection:

    Article Title: The quantitative and condition-dependent Escherichia coli proteome
    Article Snippet: Then, the samples were diluted with 100mM ammoniumbicarbonate buffer to a final sodium deoxycholate (urea) concentration of 1% (1.6M) for dataset 2 (1) samples, respectively, and further digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, Wisconsin) over night at 37°C. .. Then, the samples were diluted with 100mM ammoniumbicarbonate buffer to a final sodium deoxycholate (urea) concentration of 1% (1.6M) for dataset 2 (1) samples, respectively, and further digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, Wisconsin) over night at 37°C.

    Recombinant:

    Article Title: Mitochondrial PKM2 regulates oxidative stress-induced apoptosis by stabilizing Bcl2
    Article Snippet: An in vitro PKM2-phosphorylated recombinant Bcl2 was exhaustively acetylated with acetic anhydride and triethylamine in acetonitrile, evaporated to dryness, then resuspended in 50 mM ammonium bicarbonate buffer containing Rapigest (Waters Corp, MA, USA). .. The sample was heated to 95 °C for 10 min and then allowed to cool; 100 ng of sequencing-grade modified trypsin (Promega, Madison, WI, USA) was added.

    Isolation:

    Article Title: Cx43-Associated Secretome and Interactome Reveal Synergistic Mechanisms for Glioma Migration and MMP3 Activation
    Article Snippet: Protein digestions were conducted overnight (37°C) with sequencing-grade modified trypsin (1 μg/50 μg protein; Promega, Madison, WI, United States). .. Protein digestions were conducted overnight (37°C) with sequencing-grade modified trypsin (1 μg/50 μg protein; Promega, Madison, WI, United States).

    Article Title: Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like
    Article Snippet: In-solution tryptic digests of the samples were prepared using an enhanced filter-aided sample preparation (eFASP) method . .. Proteins were digested in the filter unit in 100 μl digestion buffer with 200 ng sequencing-grade modified trypsin (Promega) at 37 °C for 12–16 h. Peptides were isolated by centrifugation and were extracted with ethyl acetate to remove remaining deoxycholic acid . .. Three unique peptides for each protein of interest were chosen for isolation based on previous data-dependent discovery data or from online peptide databases ( www.peptideatlas.org , www.thegpm.org ).

    Article Title: Quantitative Fluorescent Labeling of Aldehyde-Tagged Proteins for Single-Molecule Imaging
    Article Snippet: Aldehyde-tagged polymerase was mixed in a 50:1 weight ratio with sequencing-grade modified trypsin (Promega) in a pH 7.8 buffer containing 100 mM ammonium bicarbonate and incubated at 37 °C overnight. .. Sample eluted off the RPLC was electrosprayed into an 11-tesla LTQ-FT Ultra hybrid mass spectrometer (Thermo).

    Article Title: Pathogen signatures activate a ubiquitinylation pathway that modulates function of the metabolic checkpoint kinase mTOR
    Article Snippet: Paragraph title: Isolation and processing of ubiquitinated proteins for proteomics analysis ... Beads were washed 6X with 8M urea buffer, resuspended in 50µl of 8M urea buffer containing 5mM DTT and incubated at 60C for 30min, followed by addition of 2-chloroacetamide to 50 mM final concentration and incubation at room temperature for 60 min. On-beads trypsin digestion were performed by diluting samples to 2M urea, adding 100ng of sequencing-grade modified trypsin (Promega) and incubating for 24hrs at 37C.

    Article Title: AMPA-receptor specific biogenesis complexes control synaptic transmission and intellectual ability
    Article Snippet: Plasma-membrane-enriched protein fractions were prepared from freshly isolated WT rat or mouse brains (pools of 10–20 animals) or from three mouse brains of CPT1c knockout animals , as previously described , and solubilized with buffers CL-47 and CL-91 (Logopharm GmbH, Germany) for 30 min on ice (at 1 mg protein per ml). .. Lanes were cut into two sections (high and low MW) and digested with sequencing-grade modified trypsin (Promega, Mannheim, Germany).

    Tandem Mass Spectroscopy:

    Article Title: Quantitative Fluorescent Labeling of Aldehyde-Tagged Proteins for Single-Molecule Imaging
    Article Snippet: Aldehyde-tagged polymerase was mixed in a 50:1 weight ratio with sequencing-grade modified trypsin (Promega) in a pH 7.8 buffer containing 100 mM ammonium bicarbonate and incubated at 37 °C overnight. .. Sample eluted off the RPLC was electrosprayed into an 11-tesla LTQ-FT Ultra hybrid mass spectrometer (Thermo).

    Article Title: A Novel Prebiotic Blend Product Prevents Irritable Bowel Syndrome in Mice by Improving Gut Microbiota and Modulating Immune Response
    Article Snippet: Then proteins were tryptic digested with 1:50 sequence-grade modified trypsin (Promega, Madison, WI, USA), and the resultant peptide mixture was labeled with iTRAQ (isobaric tags for relative and absolute quantitation). .. Then nano-HPLC (high-performance liquid chromatography)-MS (mass spectrum)/MS analysis was conducted at low pH.

    Size-exclusion Chromatography:

    Article Title: Dectin-1 Activation on Macrophages by Galectin-9 Promotes Pancreatic Carcinoma and Peritumoral Immune-Tolerance
    Article Snippet: Sequencing grade-modified trypsin (300ng; Promega, Madison, WI) was added to the dried gel pieces. .. Sequencing grade-modified trypsin (300ng; Promega, Madison, WI) was added to the dried gel pieces.

    Labeling:

    Article Title: Cx43-Associated Secretome and Interactome Reveal Synergistic Mechanisms for Glioma Migration and MMP3 Activation
    Article Snippet: Protein digestions were conducted overnight (37°C) with sequencing-grade modified trypsin (1 μg/50 μg protein; Promega, Madison, WI, United States). .. Protein digestions were conducted overnight (37°C) with sequencing-grade modified trypsin (1 μg/50 μg protein; Promega, Madison, WI, United States).

    Article Title: A Novel Prebiotic Blend Product Prevents Irritable Bowel Syndrome in Mice by Improving Gut Microbiota and Modulating Immune Response
    Article Snippet: For each sample, proteins were precipitated with ice-cold acetone, and then were re-dissolved in 100 μL TEAB (tetraethyl-ammonium bromide). .. Then proteins were tryptic digested with 1:50 sequence-grade modified trypsin (Promega, Madison, WI, USA), and the resultant peptide mixture was labeled with iTRAQ (isobaric tags for relative and absolute quantitation). .. The peptide mixture was separated at high pH, and twelve fractions were collected and each fraction dried in a vacuum concentrator for the next step.

    Article Title: The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan
    Article Snippet: Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, WI, USA) overnight at 37 °C. .. Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, WI, USA) overnight at 37 °C.

    Article Title: Systematic verification of bladder cancer-associated tissue protein biomarker candidates in clinical urine specimens
    Article Snippet: The sample was reduced by adding 12.4 μl of 50 mM Tris(2-carboxyethyl)phosphine (TCEP; final concentration, 5 mM) and incubating at 60°C for 30 min. .. The sample was then alkylated by adding 13.75 μl of 200 mM iodoacetamide (final concentration, 10 mM) and incubating at room temperature in the dark for 30 min. Sequencing-grade modified trypsin (Promega, Madison, WI, USA) was then added at a 1:20 (enzyme/substrate) ratio, and the samples were incubated at 37°C for 9 h. Tryptic peptides were stored at −20°C for subsequent dimethyl labeling. .. Tryptic peptide samples (25 μg in 100 μl of 0.1 M triethylammonium bicarbonate) were mixed with 7.5 μl of freshly prepared sodium cyanoborohydride (2 M).

    Purification:

    Article Title: Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like
    Article Snippet: Paragraph title: Targeted mass spectrometry of purified hair bundles ... Proteins were digested in the filter unit in 100 μl digestion buffer with 200 ng sequencing-grade modified trypsin (Promega) at 37 °C for 12–16 h. Peptides were isolated by centrifugation and were extracted with ethyl acetate to remove remaining deoxycholic acid .

    Lysis:

    Article Title: The quantitative and condition-dependent Escherichia coli proteome
    Article Snippet: Cells were resuspended either in 100µl lysis buffer 1 (100 mM ammoniumbicarbonate, 2% sodium deoxycholate) for the dataset 2 (see for details) or 100µl lysis buffer 2 (100 mM ammoniumbicarbonate, 8M urea, 0.1% RapiGest™) for the dataset 1. .. Then, the samples were diluted with 100mM ammoniumbicarbonate buffer to a final sodium deoxycholate (urea) concentration of 1% (1.6M) for dataset 2 (1) samples, respectively, and further digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, Wisconsin) over night at 37°C.

    Article Title: The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan
    Article Snippet: For each sample, 108 yeast cells were resuspended in 100 µl lysis buffer (2% sodium deoxycholate, 100 mM ammonium bicarbonate), sonicated for 2 × 10 s using a vial tweeter and spinned down. .. Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, WI, USA) overnight at 37 °C.

    Article Title: Comprehensive evaluation of coding region point mutations in microsatellite‐unstable colorectal cancer
    Article Snippet: The beads were then washed with 3 × 1 ml of lysis buffer and 4 × 1 ml of wash buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 50 mM NaG, 5 mM EDTA). .. Proteins were digested overnight at 37°C with sequencing‐grade modified trypsin (Promega, Madison, WI, USA).

    Article Title: The SKP1-Cullin-F-box E3 ligase βTrCP and CDK2 cooperate to control STIL abundance and centriole number
    Article Snippet: To determine relative protein abundance, 4–6 × 106 HEK 293T and 2 × 106 U2OS T-REx cells were collected and lysed in 100 µl lysis buffer (1% SDC, 0.1 M ammoniumbicarbonate, 10 mM TCEP) using strong ultra-sonication (10 min, Bioruptor, Diagnode). .. Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega) overnight at 37°C.

    SDS Page:

    Article Title: Complex formation of APP with GABAB receptors links axonal trafficking to amyloidogenic processing
    Article Snippet: Proteins were separated on SDS-PAGE gels and silver-stained. .. Lanes were cut into two sections (high and low MW) and digested with sequencing-grade modified trypsin (Promega, Mannheim, Germany).

    Article Title: AMPA-receptor specific biogenesis complexes control synaptic transmission and intellectual ability
    Article Snippet: After 2 h of incubation and two brief washes, proteins were eluted, shortly run on SDS–PAGE gels and silver-stained. .. Lanes were cut into two sections (high and low MW) and digested with sequencing-grade modified trypsin (Promega, Mannheim, Germany).

    Software:

    Article Title: A Novel Prebiotic Blend Product Prevents Irritable Bowel Syndrome in Mice by Improving Gut Microbiota and Modulating Immune Response
    Article Snippet: Then proteins were tryptic digested with 1:50 sequence-grade modified trypsin (Promega, Madison, WI, USA), and the resultant peptide mixture was labeled with iTRAQ (isobaric tags for relative and absolute quantitation). .. Then nano-HPLC (high-performance liquid chromatography)-MS (mass spectrum)/MS analysis was conducted at low pH.

    Sample Prep:

    Article Title: The quantitative and condition-dependent Escherichia coli proteome
    Article Snippet: Paragraph title: Sample preparation ... Then, the samples were diluted with 100mM ammoniumbicarbonate buffer to a final sodium deoxycholate (urea) concentration of 1% (1.6M) for dataset 2 (1) samples, respectively, and further digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, Wisconsin) over night at 37°C.

    Article Title: Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like
    Article Snippet: In-solution tryptic digests of the samples were prepared using an enhanced filter-aided sample preparation (eFASP) method . .. Proteins were digested in the filter unit in 100 μl digestion buffer with 200 ng sequencing-grade modified trypsin (Promega) at 37 °C for 12–16 h. Peptides were isolated by centrifugation and were extracted with ethyl acetate to remove remaining deoxycholic acid .

    Article Title: The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan
    Article Snippet: Paragraph title: Sample preparation ... Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, WI, USA) overnight at 37 °C.

    Article Title: The SKP1-Cullin-F-box E3 ligase βTrCP and CDK2 cooperate to control STIL abundance and centriole number
    Article Snippet: Paragraph title: Sample preparation for LC–MS analysis ... Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega) overnight at 37°C.

    In Vitro:

    Article Title: Mitochondrial PKM2 regulates oxidative stress-induced apoptosis by stabilizing Bcl2
    Article Snippet: An in vitro PKM2-phosphorylated recombinant Bcl2 was exhaustively acetylated with acetic anhydride and triethylamine in acetonitrile, evaporated to dryness, then resuspended in 50 mM ammonium bicarbonate buffer containing Rapigest (Waters Corp, MA, USA). .. The sample was heated to 95 °C for 10 min and then allowed to cool; 100 ng of sequencing-grade modified trypsin (Promega, Madison, WI, USA) was added.

    Quantitation Assay:

    Article Title: A Novel Prebiotic Blend Product Prevents Irritable Bowel Syndrome in Mice by Improving Gut Microbiota and Modulating Immune Response
    Article Snippet: For each sample, proteins were precipitated with ice-cold acetone, and then were re-dissolved in 100 μL TEAB (tetraethyl-ammonium bromide). .. Then proteins were tryptic digested with 1:50 sequence-grade modified trypsin (Promega, Madison, WI, USA), and the resultant peptide mixture was labeled with iTRAQ (isobaric tags for relative and absolute quantitation). .. The peptide mixture was separated at high pH, and twelve fractions were collected and each fraction dried in a vacuum concentrator for the next step.

    Knock-Out:

    Article Title: AMPA-receptor specific biogenesis complexes control synaptic transmission and intellectual ability
    Article Snippet: Plasma-membrane-enriched protein fractions were prepared from freshly isolated WT rat or mouse brains (pools of 10–20 animals) or from three mouse brains of CPT1c knockout animals , as previously described , and solubilized with buffers CL-47 and CL-91 (Logopharm GmbH, Germany) for 30 min on ice (at 1 mg protein per ml). .. Lanes were cut into two sections (high and low MW) and digested with sequencing-grade modified trypsin (Promega, Mannheim, Germany).

    Concentration Assay:

    Article Title: The quantitative and condition-dependent Escherichia coli proteome
    Article Snippet: After quenching the reaction with 12 mM N-acetyl- cysteine, the proteins were proteolyzed for 4h at 37°C using sequencing-grade Lys-C (Wako Chemicals) at 1/200 w/w. .. Then, the samples were diluted with 100mM ammoniumbicarbonate buffer to a final sodium deoxycholate (urea) concentration of 1% (1.6M) for dataset 2 (1) samples, respectively, and further digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, Wisconsin) over night at 37°C. .. The samples were acidified with 2M HCl to a final concentration of 50mM, incubated for 15min at 37°C and the precipitated detergent removed by centrifugation at 10,000g for 15min.

    Article Title: Chemoproteomic profiling of host and pathogen enzymes active in cholera
    Article Snippet: Acetone-washed pellets were air-dried and resuspended by sonication in 100 μL PBS containing 2.4 M urea and 70 mM ammonium bicarbonate, treated with 15 mM dithiothreitol, and incubated at 65 °C for 15 min. Iodoacetamide (12.5 mM final concentration) was then reacted with the samples for 30 min at rt. .. Samples were incubated with sequencing-grade modified trypsin (2 μg; Promega) and 1 mM CaCl2 and digested overnight at 37 °C.

    Article Title: Chemoproteomic profiling of host and pathogen enzymes active in cholera
    Article Snippet: The washed beads were suspended in 6 M urea in PBS (500 μL) and dithiothreitol (10 mM final concentration, diluted from a 20X stock in water) and incubated at 65 °C for 15 min. Iodoacetamide (20 mM final concentration, diluted from a 20X stock in water) was then reacted with the samples at 37 °C for 30 min. .. Following reduction and alkylation, the beads were pelleted by centrifugation (1400 × g, 4 °C, 3 min) and suspended in 200 μL of 2 M urea, 1 mM CaCl2 (diluted from a 100X stock in water), and sequencing-grade modified trypsin (2 μg; Promega) in PBS.

    Article Title: The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan
    Article Snippet: After quenching the reaction with 12 mM N-acetyl-cysteine the samples were diluted with 100 mM ammonium bicarbonate buffer to a final DOC concentration of 1%. .. Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, WI, USA) overnight at 37 °C.

    Article Title: Pathogen signatures activate a ubiquitinylation pathway that modulates function of the metabolic checkpoint kinase mTOR
    Article Snippet: Lysate supernatants from a high-speed spin (~4mls) were combined with 100µl of Streptavidin-agarose beads (Thermo scientific) pre-equilibrated in urea buffer and rotated at room temperature for 2hrs. .. Beads were washed 6X with 8M urea buffer, resuspended in 50µl of 8M urea buffer containing 5mM DTT and incubated at 60C for 30min, followed by addition of 2-chloroacetamide to 50 mM final concentration and incubation at room temperature for 60 min. On-beads trypsin digestion were performed by diluting samples to 2M urea, adding 100ng of sequencing-grade modified trypsin (Promega) and incubating for 24hrs at 37C. .. LC-MS/MS and subsequent analysis was carried out at the Yale W.M.

    Article Title: Systematic verification of bladder cancer-associated tissue protein biomarker candidates in clinical urine specimens
    Article Snippet: The sample was reduced by adding 12.4 μl of 50 mM Tris(2-carboxyethyl)phosphine (TCEP; final concentration, 5 mM) and incubating at 60°C for 30 min. .. The sample was then alkylated by adding 13.75 μl of 200 mM iodoacetamide (final concentration, 10 mM) and incubating at room temperature in the dark for 30 min. Sequencing-grade modified trypsin (Promega, Madison, WI, USA) was then added at a 1:20 (enzyme/substrate) ratio, and the samples were incubated at 37°C for 9 h. Tryptic peptides were stored at −20°C for subsequent dimethyl labeling. .. Tryptic peptide samples (25 μg in 100 μl of 0.1 M triethylammonium bicarbonate) were mixed with 7.5 μl of freshly prepared sodium cyanoborohydride (2 M).

    Article Title: The SKP1-Cullin-F-box E3 ligase βTrCP and CDK2 cooperate to control STIL abundance and centriole number
    Article Snippet: Sample aliquots containing 50 µg of total proteins were reduced for 10 min at 95°C and alkylated by adding chloroacetamide to 15 mM final concentration for 30 min at 37°C. .. Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega) overnight at 37°C.

    Liquid Chromatography with Mass Spectroscopy:

    Article Title: The quantitative and condition-dependent Escherichia coli proteome
    Article Snippet: Then, the samples were diluted with 100mM ammoniumbicarbonate buffer to a final sodium deoxycholate (urea) concentration of 1% (1.6M) for dataset 2 (1) samples, respectively, and further digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, Wisconsin) over night at 37°C. .. Then, the samples were diluted with 100mM ammoniumbicarbonate buffer to a final sodium deoxycholate (urea) concentration of 1% (1.6M) for dataset 2 (1) samples, respectively, and further digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega, Madison, Wisconsin) over night at 37°C.

    Article Title: Type VI secretion system sheath inter‐subunit interactions modulate its contraction
    Article Snippet: Proteins were digested using sequencing‐grade modified trypsin (1/250, w/w; Promega, USA) overnight at 37°C. .. After digestion, the samples were supplemented with trifluoroacetic acid to a final concentration of 1%.

    Article Title: The SKP1-Cullin-F-box E3 ligase βTrCP and CDK2 cooperate to control STIL abundance and centriole number
    Article Snippet: Paragraph title: Sample preparation for LC–MS analysis ... Proteins were digested by incubation with sequencing-grade modified trypsin (1/50, w/w; Promega) overnight at 37°C.

    Staining:

    Article Title: Dectin-1 Activation on Macrophages by Galectin-9 Promotes Pancreatic Carcinoma and Peritumoral Immune-Tolerance
    Article Snippet: Sequencing grade-modified trypsin (300ng; Promega, Madison, WI) was added to the dried gel pieces. .. Sequencing grade-modified trypsin (300ng; Promega, Madison, WI) was added to the dried gel pieces.

    Affinity Purification:

    Article Title: Comprehensive evaluation of coding region point mutations in microsatellite‐unstable colorectal cancer
    Article Snippet: Paragraph title: Interactor affinity purification ... Proteins were digested overnight at 37°C with sequencing‐grade modified trypsin (Promega, Madison, WI, USA).

    Article Title: AMPA-receptor specific biogenesis complexes control synaptic transmission and intellectual ability
    Article Snippet: Lanes were cut into two sections (high and low MW) and digested with sequencing-grade modified trypsin (Promega, Mannheim, Germany). .. Lanes were cut into two sections (high and low MW) and digested with sequencing-grade modified trypsin (Promega, Mannheim, Germany).

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    Promega sequencing grade modified trypsin
    Sequencing Grade Modified Trypsin, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 559 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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