LFQ_TurboID-STING_DMXAA_Biotin_Antibody_RAW2647
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus Musculus (mouse)
SUBMITTER: Hidetaka Kosako
PROVIDER: PXD017044 | JPOST Repository | Sun Jan 10 00:00:00 GMT 2021
REPOSITORIES: jPOST
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Project description:The STING-null RAW264.7 cells expressing TurboID-STING were treated with DMSO or 100 μg/ml DMXAA, and 500 µM biotin was added to the medium. After 1-h incubation, the cells were lysed in 6 M guanidine-HCl containing 100 mM Tris-HCl (pH 8.0) and 2 mM DTT. The lysates were dissolved by heating and sonication, and centrifuged at 20,000 g for 15 min at 4ºC. The supernatants were recovered, followed by reduction in 5 mM DTT at room temperature for 30 min and alkylation in 27.5 mM iodoacetamide at room temperature for 30 min in the dark. Proteins were purified by methanol/chloroform precipitation and solubilized by 0.1% RapiGest SF in 50 mM triethylammonium bicarbonate. After repeated sonication and vortex, the proteins were digested with 20 µg trypsin at 37ºC overnight. The resultant peptide solutions were captured on MagCapture Tamavidin 2-REV magnetic beads in the presence of 1 mg/ml Pefabloc SC during 16 h of incubation at 4ºC. After washing with TBS five times, the biotinylated peptides were eluted with 100 µL of 2 mM biotin at 95ºC twice. The combined eluates were desalted using GL-Tip SDB, evaporated in a SpeedVac concentrato, and redissolved in 0.1% TFA.
2023-04-17 | PXD017042 | JPOST Repository
Project description:The STING-null RAW264.7 cells expressing TurboID-STING were treated with DMSO or 30 μg/ml DMXAA, and 500 µM biotin was added to the medium. After 1-h incubation, the cells were lysed in 6 M guanidine-HCl containing 100 mM HEPES-NaOH (pH 7.5), 10 mM TCEP, and 40 mM CAA. The lysates were dissolved by heating and sonication, and centrifuged at 20,000 g for 15 min at 4ºC. The supernatants were recovered, and proteins were purified by methanol/chloroform precipitation and solubilized by 0.1% RapiGest SF in 50 mM triethylammonium bicarbonate. After repeated sonication and vortex, the proteins were digested with trypsin at 37ºC overnight. The resultant peptide solutions were captured on MagCapture HP Tamavidin 2-REV magnetic beads in the presence of 1 mg/ml Pefabloc SC during 3 h of incubation at 4ºC. After washing with TBS five times, the biotinylated peptides were eluted with 100 µL of 1 mM biotin at 95ºC twice. The combined eluates were desalted using GL-Tip SDB, evaporated in a SpeedVac concentrato, and redissolved in 0.1% TFA and 3% ACN.
2020-06-17 | PXD018802 | JPOST Repository
Project description:The STING-null RAW264.7 cells expressing TurboID-STING were treated with DMSO or 30 μg/ml DMXAA, and 500 µM biotin was added to the medium. After 1-h incubation, the cells were lysed in 6 M guanidine-HCl containing 100 mM HEPES-NaOH (pH 7.5), 10 mM TCEP, and 40 mM CAA. The lysates were dissolved by heating and sonication, and centrifuged at 20,000 g for 15 min at 4ºC. The supernatants were recovered, and proteins were purified by methanol/chloroform precipitation and solubilized by 0.1% RapiGest SF in 50 mM triethylammonium bicarbonate. After repeated sonication and vortex, the proteins were digested with trypsin at 37ºC overnight. The resultant peptide solutions were captured on anti-biotin (A7C2A) rabbit monoclonal antibody-conjugated beads in the presence of 1 mg/ml Pefabloc SC during 3 h of incubation at 4ºC. After washing with TBS four times, and with ultrapure water twice, the biotinylated peptides were eluted with 100 µL of 0.2% TFA and 80% ACN twice. The combined eluates were desalted using GL-Tip SDB, evaporated in a SpeedVac concentrator, and redissolved in 0.1% TFA and 3% ACN.
2020-06-17 | PXD018803 | JPOST Repository
Project description:Sperm isolated from WT mice and PITHD1-deficient mice in triplicate were lysed in 6 M guanidine-HCl containing 100 mM Tris-HCl, pH 8.0, and 2 mM DTT. The lysates were dissolved by heating and sonication, followed by centrifugation at 20,000 × g for 15 min at 4 °C. The supernatants containing 100 µg protein each were reduced in 5 mM DTT at room temperature for 30 min and alkylated in 27.5 mM iodoacetamide at room temperature for 30 min in the dark. Proteins were purified by methanol/chloroform precipitation and solubilized with 25 µL of 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate buffer. After repeated sonication and vortexing, the proteins were digested with 1 μg of trypsin/Lys-C mix (Promega) for 16 h at 37 °C. Approximately 25 µg of peptides for each sample was labeled with 0.2 mg of TMT6plex reagents for 1 h at room temperature. After the reaction was quenched with hydroxylamine, all the TMT-labeled samples were pooled, acidified with trifluoroacetic acid (TFA), and fractionated using a Pierce High pH Reversed-phase Peptide Fractionation Kit (Thermo Fisher). Nine fractions were collected using 5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, and 50% acetonitrile (ACN) in 0.1% triethylamine. Each fraction was evaporated in a SpeedVac concentrator and dissolved in 0.1% TFA.
2020-01-10 | PXD015347 | JPOST Repository
Project description:Bacteria was grown at 30C in 3 different conditions, i.e. SYN (syngas and minimal medium- ATCC no 1789), AC (0.3% acetate in minimal medium- ATCC no 1789) and TSB (tryptic soy broth). After harvesting by centrifugation, O. carboxidovorans pellets (1g) were lysed in 100 mM Tris-Cl pH 8.0, 2% Triton X-100, 2.6 mg/ml sodium azide, 8 mM PMSF by sonication on ice (4 pulses of 15 s duration each). For each condition of growth 4 samples (1g pellets) were separately treated (i.e. lysed and processed further). The supernatants were treated with 50% cold TCA, and the precipitated protein washed with acetone. The pellets were resuspended in solubilization buffer (7M urea, 20 mM tris-Cl, pH 8.0, 5 mM EDTA, 5 mM MgCl2, 4% CHAPS), and protein concentration was determined using the Plus One 2-D Quant Kit (Amersham) following the manufacturers instructions. Protein samples from each treatment were stored at -80 C. One hundred micrograms of each protein sample was resuspended in 0.1 M ammonium bicarbonate, 5% HPLC grade ACN, reduced in 5 mM DTT (65 C, 5 min), alkylated in 10 mM iodoacetamide (30 C, 30 min), and then trypsin digested until there was no visible pellet (1:50 w/w 37 C, 16 h). Peptides were desalted using a peptide microtrap (Michrom BioResources, Auburn, CA) and eluted using a 0.1% TFA, 95% ACN solution. Desalted peptides were dried in a vacuum centrifuge and resuspended in 20 ?l of 0.1% formic acid. Peptides were separated by strong cation exchange (SCX) liquid chromatography (LC) followed by reverse phase (RP) LC coupled directly in line with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). 2DLC ESI MS/MS was done exactly as described (1). All searches were done using TurboSEQUEST (Bioworks Browser 3.2; Thermo Electron). Mass spectra and tandem mass spectra were searched against all annotated proteins from the strain OM5 including all the annotated plasmid-encoded proteins. Cysteine carbamidomethylation and methionine oxidation (single and double) were included in the search strategy. We used the reverse database functionality in Bioworks 3.2 and searched MS2 data against a reversed OM5 database using identical search criteria.
2012-05-23 | PRD000150 | Pride
Project description:MicroPoly(A)Pure kits (Ambion) were used to prepare mRNA from P. salmonis-infected and control Atlantic salmon head kidney. Head kidney mRNA samples were prepared from pooled (n = 10) infected or control kidney. Approximately 200 ng of infected or control head kidney mRNA was used as template in aRNA synthesis reactions. Two infected head kidney samples were pooled to yield 8.9 ug of aRNA, and a single control head kidney sample contained 8.4 ug of aRNA. Infected and control head kidney aRNA samples were visualized on agarose gels to check quality and quantity (data not shown). Except for the amounts of aRNA and reverse transcription (RT) primer used, head kidney target labeling reactions and microarray hybridizations were identical. Head kidney target syntheses used 2 ug of aRNA and 2 ug of random primers (Roche). RT primers were mixed with aRNA samples, and nuclease-free H2O (Invitrogen) was added to make 10 ul total volume. Samples were incubated at 70 degrees C for 4 min, then placed on ice. RT reactions included 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.05 mM Cy3-dUTP or Cy5-dUTP (Amersham), 40U RNAguard ribonuclease inhibitor (Amersham), and 400U Superscript II Rnase H- reverse transcriptase (Invitrogen). RT reactions were incubated at 42 degrees C for 2.5 h, followed by addition of 0.025U RNase A (Sigma) and 1.5U RNase H (New England BioLabs) and incubation at 37 degrees C for 30 min. Labeled targets were cleaned using the Qiaquick PCR purification kit (Qiagen) and precipitated, with 300 mM sodium acetate, 1 ul of 20 ug/ul glycogen (Invitrogen), and 2.5 volumes of 100% ethanol, at -20 degrees C overnight. Each labeled target was recovered by centrifugation (14,000 rpm, 4 degrees C, 1 h), washed in 70% ethanol, air-dried, and resuspended in 60 ul of hybridization buffer: 50% deionized formamide, 5X SSC, 0.1% SDS, 5 ul of 5 ug/ul oligo dT (5’T18[Q-N]3’), 5 ul of 2 ug/ul BSA (Pierce), and 2 ul of 10 ug/ul sonicated human placental DNA (Sigma). Targets were incubated at 96 degrees C for 3 min, and then at 65 degrees C until applied to microarrays. Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95 degrees C MilliQ H2O, and drying by centrifugation (2000 rpm, 5 min, in 50 ml conical tube). Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 45 degrees C water bath for 16 h. Coverslips were floated off in 45 degrees C (1X SSC, 0.2% SDS), and arrays were washed 1 X 10 min in 45 degrees C (1X SSC, 0.2% SDS), 3 X 4 min in room temperature (0.1X SSC, 0.1% SDS), and 4 X 4 min in room temperature 0.1X SSC. Slides were dried by centrifugation as before. Fluorescent images of hybridized arrays were acquired immediately at 10 um resolution using ScanArray Express (PerkinElmer). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm respectively, and the same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in a study (PMT 75 Cy3, PMT 63 or 64 Cy5 for head kidney study). Fluorescent intensity data were extracted from TIF images using Imagene 5.5 software (Biodiscovery). Keywords: other
2004-02-11 | GSE1031 | GEO
Project description:MicroPoly(A)Pure kits (Ambion) were used to prepare mRNA from P. salmonis-infected and control Atlantic salmon macrophages. Infected and control (24 h incubation) macrophage mRNA samples were subjected to two rounds of amplification using the MessageAmp aRNA kit (Ambion) and manufacturer's instructions. Approximately 400 ng of macrophage mRNA was used as template in the first round of amplification, yielding 8.1 ug of infected macrophage aRNA and 7.8 ug of control macrophage aRNA. Approximately 1 ug of first-round macrophage aRNA was used as template in the second round of amplification, yielding 81.2 ug of infected macrophage aRNA and 83.3 ug of control macrophage aRNA. Infected and control macrophage aRNA samples were visualized on agarose gels to check quality and quantity. Except for the amounts of aRNA and reverse transcription (RT) primer used, macrophage target labeling reactions and microarray hybridizations were identical. Macrophage target syntheses used 5 ug of second-round aRNA and 5 ug of random primers (Roche). RT primers were mixed with aRNA samples, and nuclease-free H2O (Invitrogen) was added to make 10 ul total volume. Samples were incubated at 70 degrees C 4 min, then placed on ice. RT reactions included 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.05 mM Cy3-dUTP or Cy5-dUTP (Amersham), 40U RNAguard ribonuclease inhibitor (Amersham), and 400U Superscript II Rnase H- reverse transcriptase (Invitrogen). RT reactions were incubated at 42 degrees C for 2.5 h, followed by addition of 0.025U RNase A (Sigma) and 1.5U RNase H (New England BioLabs) and incubation at 37 degrees C for 30 min. Labeled targets were cleaned using the Qiaquick PCR purification kit (Qiagen) and precipitated, with 300 mM sodium acetate, 1 ul of 20 ug/ul glycogen (Invitrogen), and 2.5 volumes of 100% ethanol, at -20 degrees C overnight. Each labeled target was recovered by centrifugation (14,000 rpm, 4 degrees C, 1 h), washed in 70% ethanol, air-dried, and resuspended in 60 ul of hybridization buffer: 50% deionized formamide, 5X SSC, 0.1% SDS, 5 ul of 5 ug/ul oligo dT (5'T18[Q-N]3'), 5 ul of 2 ug/ul BSA (Pierce), and 2 ul of 10 ug/ul sonicated human placental DNA (Sigma). Targets were incubated at 96 degrees C for 3 min, and then at 65 degrees C until applied to microarrays. Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95 degrees C MilliQ H2O, and drying by centrifugation (2000 rpm, 5 min, in 50 ml conical tube). Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 45 degrees C water bath for 16 h. Coverslips were floated off in 45 degrees C (1X SSC, 0.2% SDS), and arrays were washed 1 X 10 min in 45 degrees C (1X SSC, 0.2% SDS), 3 X 4 min in room temperature (0.1X SSC, 0.1% SDS), and 4 X 4 min in room temperature 0.1X SSC. Slides were dried by centrifugation as before. Fluorescent images of hybridized arrays were acquired immediately at 10 um resolution using ScanArray Express (PerkinElmer). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm respectively, and the same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in a study (PMT 72 Cy3, PMT 63 or 64 Cy5 for macrophage study). Fluorescent intensity data were extracted from TIF images using Imagene 5.5 software (Biodiscovery). Keywords: other
2004-02-11 | GSE1012 | GEO
Project description:Biochemical purification: Five grams of adult flies or 2.5 g of embryos were used in each affinity purification. Flies or embryos were suspended in 15 ml of buffer B (buffer A plus 1.5 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonylfluoride [PMSF], 0.5 5g/ml leupeptin, 0.8 5g/ml pepstatin, 20 U/ml DNase I, 100 U/ml RNasin [Promega], and 0.2 mg/ml heparin) in a mortar filled with liquid nitrogen and ground with a pestle to a fine powder. The powder was transferred to a glass-dounce, thawed and dounced until the pestle reached the bottom. The suspension was centrifuged twice at 40C and 10,000 g for 10 min. The fat layer on top was aspirated off after each centrifugation. Cleared extract (12.5 ml) was incubated with 600 5l of a slurry (50% [v/v]) of IgG-agarose beads (Sigma) for 90 min at 4C. The beads were washed once with buffer B for 15 min at 4C, and three times for 15 min at 4C with buffer C (20 mM TrisHCl [pH 8.0], 150 mM NaCl, 1 mM EDTA [pH 8.0], 10% glycerol, 0.01% NP-40, 1 mM DTT, 10 U/ml RNasin). PumHD was released from beads by incubation with 150 Units of AcTEV protease (Invitrogen) for 2 hr at room temperature (RT). RNA was isolated from extracts and from the TEV eluates with TRIZOL reagent (Invitrogen) followed by RNeasy Mini-Kit (Qiagen) purification according to the manufacturer's instructions. Microarray analysis: A pool of four control cDNAs (2 ng of each flp, gal4, lacZ and -Gal) was added to each RNA sample prior to labeling as controls for the labeling procedure. Total RNA (12 5g) derived from the extract and 300 ng or 30% of the affinity-isolated RNA were labeled with Cy3 and Cy5 fluorescent dyes, respectively, following cDNA synthesis with amino-allyl dUTP in addition to the four natural dNTPs using a 1:1 mixture of oligo(dT) and random nonamer primers. The Cy3- and Cy5-labeled cDNA samples were mixed and hybridized to Drosophila DNA microarrays. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Keywords: RNA IP
2006-02-28 | GSE3582 | GEO
Project description:Proteome quantification using TMT reagents and off-line basic peptide fractionation was done as described previously in Grand, 2021, with the exception that the isolated nuclei were used as a starting material rather than whole cells to enable detection of transcription factors which are generally less abundant proteins in the cell, and that nine channels from a TMTpro16 reagent kit were used. Briefly, mESCs, NGN2 24h induced cells and MyoD1 24h induced cells were harvested in triplicates of 10 million per sample. Nuclei were isolated by suspending in 4 ml of nuclear extraction buffer (10 mM HEPES pH 7.9, 10 mM KCl, 10 mM EDTA, 0.5% NP-40, 1 mM DTT and complete protease inhibitor (Roche)) and incubating on ice for 10 min with vortexing every 2 minutes. Nuclei were collected by centrifugation (800g, 10 min, 4 °C), washed with ice cold PBS and suspended in 1% sodium deoxycholate in 50 mM HEPES buffer (pH 8.5). The nuclear lysate was disrupted with sonication. Proteins were reduced, alkylated and Lys-C/trypsin digested. Fifty micrograms of peptides from each biological replicate were labelled using channels 128C-131C from a TMT 16plex isobaric labelling reagents kit (Lot# VJ313476, Thermo Fisher Scientific). Dried TMT-labeled peptides were dissolved in 20 μL of 10 mM ammonium formate (pH 10, high pH Buffer A), and high-pH reversed-phase chromatography was employed using an Agilent 1100 HPLC system with an autosampler, a YMC Triart C18 0.5 x 250 mm column, and a fraction collector. The peptides were separated using the following binary buffer system: high-pH Buffer A (20 mM ammonium formate in water, pH 10) and high-pH Buffer B (20 mM ammonium formate pH 10 in 90% acetonitrile) at a flow rate of 12 μL/minute. The gradient duration was a total of 115 min, with mobile phase compositions in %B as follows: 0-5 min at 2%-15%, 5-15 min at 15%-25%, 15-80 min at 25%-45%, 80-85 min at 45%-65%, 85-95 min at 65%-100%, 95-100 min at 100%, 100-101 min at 100%-2%, and 101-115 min at 2%, resulting in 48 fractions after sample concatenation. Samples were acidified with 1% TFA and dried in a speed vac. Peptides reconstituted in 2% acetonitrile, 0.1% formic acid were on-line separated on a C18 50 cm μPAC column using a EASYnLC-1000 system (all Thermo Fisher Scientific) mounted on a Digital PicoView nanospray source (New Objective), connected to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Peptide elution was achieved using a linear gradient of increasing concentration of acetonitrile in 0.1% formic acid at a flow rate of 500 nl/min: 0-2 min: 2%, 2-8 min: 2-6%, 8-89 min 6-22%, 89-107 min: 22-40%, 107-111 min: 40-80%, 111-115 min: 80% (washing), 115-116 min: 80-2%, 116-120 min: 2% (equilibration). For MS data acquisition in a data-dependent mode, ions for survey MS1 scans were recorded in profile mode in the Orbitrap detector at a resolution of 120,000 in the range of 400-1400 m/z every 3 seconds for a maximum of 100 ms to reach the normalized AGC target of 50%. The most abundant precursors were selected in the quadrupole within an isolation window of 0.7 m/z for MS2 HCD fragmentation based on advanced peak determination, monoisotopic peak determination set to peptides, within charge states 2-8, dynamic exclusion of 60 s within +/-10 ppm tolerance for isotopes and different charge states, with a minimum intensity of 5e4, with normalized collision energy 34% for a maximum injection time of 200 ms to reach the normalized AGC target of 80%. MS2 HCD spectra were and recorded in the “normal” range, starting at 100 m/z at 50,000 resolution in the Orbitrap analyzer in centroid mode.
2025-08-21 | PXD054143 | Pride
Project description:Transcriptomic study of lpp2 KO mutant in response to abcsisic acid - Fully developed rosette leaves were immersed for 1 min in a solution of 10-5 M ABA with 0.1% DMSO and 0.05%silwet. RNA are extracted after a 3h incubation. This treatment is compared to control (1 min immersion in 0.1% DMSO and 0.05%silwet) then 3h incubation. Keywords: treated vs untreated comparison
2009-07-01 | GSE14446 | GEO