An effective response to respiratory inhibition by a Pseudomonas aeruginosa excreted quinoline promotes Staphylococcus aureus fitness and survival in co-culture
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive Plus
ORGANISM(S): Staphylococcus Aureus Subsp. Aureus Usa300_lac
SUBMITTER: 
Jeffrey M. Boyd
PROVIDER: MSV000099005 | MassIVE |
REPOSITORIES: MassIVE
Similar Datasets
Project description:Triplicate cultures of S. aureus USA300_LAC were cultured overnight in TSB before diluting to 0.1 (A600) in 5 mL of TSB in 25 mL culture tubes. Cells were grown for an additional 8 hours with 5 ug mL-1 Pseudomonas aeruginosa HQNO or vehicle control, after which 1 mL of each sample was centrifuged at 13,000g for 1 minute and washed once with PBS. Cell pellets were resuspended in 1 mL of an ice-cold 2:2:1 solution of Methanol:Acetonitrile:H2O. Harvested cells were lysed in a FastPrep homogenizer (MP Biomedicals) with 600 uL of 0.1 mm lysing matrix beads (MP Biomedicals) (2 cycles, 40 sec, 6.0 m sec-1). Samples were incubated on ice for 5 minutes before centrifuging twice at 13,000g for 2 minutes at 4C. A total of 850 uL of the supernatant was added to a spin filter and centrifuged again at 4C at 13,000g for 15 minutes. The samples were frozen at -80C until transferred to the metabolomics core of the Rutgers Cancer Institute of New Jersey for analysis.
| MSV000099005 | GNPS
Project description:We have performred RNA-seq analysis of WT Mtb H37Rv, ∆phoP Mtb and H37Rv-pde (WT Mtb H37Rv overexpressing gene Rv0805 which codes for a phosphodiesterase), to look for the similarity in ∆phoP Mtb and H37Rv-pde transcriptome. RNA was extracted from exponentially growing mycobacterial cells in Middlebrook 7H9 media. Briefly, 25 ml of bacterial culture was grown to mid-log phase (OD600= 0.4 to 0.6) and combined with 40 ml of 5 M guanidinium thiocyanate solution containing 1% β-mercaptoethanol and 0.5% Tween 80. Cells were pelleted by centrifugation, and lysed by re-suspending in 1 ml Trizol (Ambion) in the presence of Lysing Matrix B (100 µm silica beads; MP Bio) using a FastPrep-24 bead beater (MP Bio) at a speed setting of 6.0 for 30 seconds. The procedure was repeated for 2-3 cycles with incubation on ice in between pulses. Next, cell lysates were centrifuged at 13000 rpm for 10 minutes; supernatant was collected and processed for RNA isolation using Direct-ZolTM RNA isolation kit (ZYMO) as per manufacturer’s recommendation. Following extraction, RNA was treated with DNAse I (Promega) to degrade contaminating DNA, and integrity was assessed using a Nanodrop (ND-1000, Spectrophotometer). RNA samples were further checked for intactness of 23S and 16S rRNA using formaldehyde-agarose gel electrophoresis, and Qubit fluoremeter (Invitrogen). RNA integrity was checked using Agilent 2200 Tape Station system (Agilent Technologies). Library construction, RNA-sequencing and data analysis have been carried out by Agrigenome Labs Private Limited (Cochin), India.
2022-04-22 | GSE173225 | GEO
Project description:Approximately 1 x 10^8 GFP-HA-KERP2 expressing Entamoeba histolytica cells were cross-linked in 0.5% formaldehyde (in PBS) for 10 minutes at room temperature. Cross-linking was quenched with 125 mM glycine for 5 minutes. Cells were washed once with PBS and subjected to nuclear fractionation using nuclei isolation buffer (10 mM Hepes-KOH pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 2% NP-40, 1 uM E-64, 0.5 mM PMSF, and 1x cOmplete protease inhibitor cocktail). Nuclear pellets were resuspended in sonication buffer (50 mM Tris-HCl pH 8.0, 140 mM NaCl2, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 5 mM DTT, 1 uM E-64, 0.5 mM PMSF, 1x cOmplete) and sonicated using a Covaris instrument. Sonicated samples were centrifuged at 16000 x g for 20 minutes. A 200 uL aliquot of supernatant was transferred to a 1.5 mL Protein LoBind tube. Remaining sample was stored as input control. For immunoprecipitation, 1 mL of ChIP dilution buffer (16.7 mM Tris-HCl pH 8.0, 0.01% SDS, 1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl, 0.5% BSA, 1x cOmplete) containing 50 uL of GFP(ab290)-antibody conjugated Dynabeads was added and rotated at 4 C for 12 hours. Beads were washed five times with low-salt wash buffer (20 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl), followed by two washes with high-salt wash buffer (same as above, but 500 mM NaCl). DNA-protein complexes were eluted with 200 uL of ChIP elution buffer (10 mM Tris-HCl pH 8.0, 1% SDS, 5 mM EDTA, 300 mM NaCl) at room temperature for 20 minutes. Input samples were thawed and diluted with 190 uL of elution buffer. All samples were incubated at 65 C for 8 hours to reverse cross-links, followed by RNase A treatment (1 uL) at 37 C for 1 hour and Proteinase K digestion (5 uL) at 55 C for 2 hours. DNA was purified by phenol/chloroform extraction with 1 uL glycogen as carrier, followed by ethanol precipitation overnight at -30 C. DNA pellets were washed with 80% ethanol, air-dried, and resuspended in 20 uL TE (ChIP) or 40 uL TE (input). DNA concentrations were quantified using a Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific). Purified DNA was submitted to Rhelixa for ChIP-seq.
2026-03-02 | GSE292714 | GEO
Project description:Patients received standardized coartem therapy. Briefly, each pill contains 20 mg of artemether and 120 mg of lumefantrine. The dose and total coarse of tablets is based on the patients body weight. Blood was drawn from patients prior to anti-malarial treatment (day 0) and after treatment (day42 for children and day 28 for adults). Peripheral Blood Mononuclear Cells were isolated from all patients. Briefly, for PBMC isolation, whole blood from was diluted 1:2 in sterile PBS and overlaid on Ficoll Paque-PLUS in 50 mL conical tubes and centrifuged 800xg for 15 minutes at 25°C with no brake. Resulting buffy coats were collected and washed twice in RPMI 1640 medium, and then cells were treated with Red Blood Cell Lysis Buffer for 3 minutes at room temperature. Cells were washed in RPMI and counted on a hemacytometer. Monocytes were purified from PBMC using a miltenyi Pan Monocyte Isolation negative selection kit according to the manufacturer's instructions. For each sample. 50,000 viable cells were pelleted and lysed with cold ATAC-Resuspension Buffer (RSB) containing 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin and incubated in ice for 3 minutes. The lysaste was washed with 1 ml of cold ATAC-RSB containing 0.1% Tween-20 but NO NP40 or digitonin. Nuclei were pelleted at 500 RCF for 10 min at 4 degrees celcius. The supernatant was discarded and the pellet was resuspended in 50 uL of transposition mixture (25 ul 2x TD buffer, 2.5 ul transposase (100nM final), 16.5 ul PBS,0.5 ul 1% digitonin, 0.5 ul 10% Tween-20, 5 ul H2O). The reaction was incubated for 30 minutes in a thermomixer with 1000 RPM mixing. Zymo DNA Clean and concentrator kit (cat# D4014) was used as a cleanup step. The DNA was amplified at 98 degrees C for 30 seconds, then 13 cycles of: 98 degrees C for 10 seconds, 63 degrees C for 30 seconds, 72 degrees C for 1 minute.The library was evaluated in an Agilent TapeStation system and the DNA concentration evaluated by Qubit. Sequencing was performed on Illumina Nextseq 500 with Buffer cartridge v2: Ref: 15057941, High output reagent cartridge v2: Ref: 15057934, NextSeq accessory Box v2: Ref: 15058251, and High output Flow Cell cartridge v2.5: Ref: 20022408
2022-04-24 | E-MTAB-11643 | biostudies-arrayexpress
Project description:We have performred RNA-seq analysis of 2851 Knockdown to investigate the role of this gene in maintaning the homoeostasis of bacterium. RNA was extracted from exponentially growing mycobacterial cells in Middlebrook 7H9 media. For cells with ATC and without ATC, re-suspended in media , and finally the cells were grown further for 0.4-0.6OD at 37°C. After incubation cells were combined with 40 ml of 5 M guanidinium thiocyanate solution containing 1% β-mercaptoethanol and 0.5% Tween 80. Cells were pelleted by centrifugation, and lysed by re-suspending in 1 ml Trizol (Ambion) in the presence of Lysing Matrix B (100 µm silica beads; MP Bio) using a FastPrep-24 bead beater (MP Bio) at a speed setting of 6.0 for 30 seconds. The procedure was repeated for 2-3 cycles with incubation on ice in between pulses. Next, cell lysates were centrifuged at 13000 rpm for 10 minutes; supernatant was collected and processed for RNA isolation using Direct-ZolTM RNA isolation kit (ZYMO) as per manufacturer’s recommendation. Following extraction, RNA was treated with DNAse I (Promega) to degrade contaminating DNA, and integrity was assessed using a Nanodrop (ND-1000, Spectrophotometer). RNA samples were further checked for intactness of 23S and 16S rRNA using formaldehyde-agarose gel electrophoresis, and Qubit fluoremeter (Invitrogen). RNA integrity was checked using Agilent 2200 Tape Station system (Agilent Technologies). Library construction, RNA-sequencing and data analysis have been carried out by Medgenome Labs Private Limited , India. The main purpose of this study is to understand how mycobacteria can sense acidic stress conditions and mount an appropriate stress response.
2025-08-15 | GSE298381 | GEO
Project description:We have performred RNA-seq analysis of WT and ∆phoR Mtb H37Rv under normal and acidic stress, to investigate the role of PhoR in maintaning the pH homoeostasis of bacterium. RNA was extracted from exponentially growing mycobacterial cells in Middlebrook 7H9 media. For acid stress, cells at OD600 of 0.6 were pelleted, washed twice with 7H9 medium buffered with 100 mM MOPS or 2N HCl for pH 7.0 or pH 4.5, respectively, re-suspended in media of indicated pH, and finally the cells were grown further for 2 hours at 37°C. After incubation cells were combined with 40 ml of 5 M guanidinium thiocyanate solution containing 1% β-mercaptoethanol and 0.5% Tween 80. Cells were pelleted by centrifugation, and lysed by re-suspending in 1 ml Trizol (Ambion) in the presence of Lysing Matrix B (100 µm silica beads; MP Bio) using a FastPrep-24 bead beater (MP Bio) at a speed setting of 6.0 for 30 seconds. The procedure was repeated for 2-3 cycles with incubation on ice in between pulses. Next, cell lysates were centrifuged at 13000 rpm for 10 minutes; supernatant was collected and processed for RNA isolation using Direct-ZolTM RNA isolation kit (ZYMO) as per manufacturer’s recommendation. Following extraction, RNA was treated with DNAse I (Promega) to degrade contaminating DNA, and integrity was assessed using a Nanodrop (ND-1000, Spectrophotometer). RNA samples were further checked for intactness of 23S and 16S rRNA using formaldehyde-agarose gel electrophoresis, and Qubit fluoremeter (Invitrogen). RNA integrity was checked using Agilent 2200 Tape Station system (Agilent Technologies). Library construction, RNA-sequencing and data analysis have been carried out by Agrigenome Labs Private Limited (Cochin), India. The main purpose of this study is to understand how mycobacteria can sense acidic stress conditions and mount an appropriate stress response.
2022-07-15 | GSE180161 | GEO
Project description:We have performred RNA-seq analysis of WT under gycerol and acetate at acidic pH to investigate the role of PhoP in maintaning the pH homoeostasis of bacterium. RNA was extracted from exponentially growing mycobacterial cells in Middlebrook 7H9 media. For acid stress, cells at OD600 of 0.6 were pelleted, washed twice with 7H9 medium buffered with 100 mM MOPS or 2N HCl for pH 7.0 or pH 4.5, respectively, re-suspended in media of indicated pH, and finally the cells were grown further for 2 hours at 37°C. After incubation cells were combined with 40 ml of 5 M guanidinium thiocyanate solution containing 1% β-mercaptoethanol and 0.5% Tween 80. Cells were pelleted by centrifugation, and lysed by re-suspending in 1 ml Trizol (Ambion) in the presence of Lysing Matrix B (100 µm silica beads; MP Bio) using a FastPrep-24 bead beater (MP Bio) at a speed setting of 6.0 for 30 seconds. The procedure was repeated for 2-3 cycles with incubation on ice in between pulses. Next, cell lysates were centrifuged at 13000 rpm for 10 minutes; supernatant was collected and processed for RNA isolation using Direct-ZolTM RNA isolation kit (ZYMO) as per manufacturer’s recommendation. Following extraction, RNA was treated with DNAse I (Promega) to degrade contaminating DNA, and integrity was assessed using a Nanodrop (ND-1000, Spectrophotometer). RNA samples were further checked for intactness of 23S and 16S rRNA using formaldehyde-agarose gel electrophoresis, and Qubit fluoremeter (Invitrogen). RNA integrity was checked using Agilent 2200 Tape Station system (Agilent Technologies). Library construction, RNA-sequencing and data analysis have been carried out by Medgenome Labs Private Limited , India. The main purpose of this study is to understand how mycobacteria can sense acidic stress conditions and mount an appropriate stress response.
2025-08-15 | GSE297253 | GEO
Project description:A mouse hepatoma cell line (Hepa1-6 cells, ATCC) was grown in T-25 tissue culture flasks (Corning) at 37ºC in 5% CO2 under two conditions. The first condition was the control, which contained DMEM medium (Gibco BRL) with 25 mM glucose, 4 mM glutamine, 1% penicillin-streptomycin (Sigma), and 10% CPSR-3 (Sigma). The second condition was identical to the first, however the glutamine concentration was altered periodically. Once the cells had grown to confluency, the experiment began at 9:30 PM by exchanging the medium in all flasks. The control flasks received the same medium, containing 4 mM glutamine, while the experimental flasks received identical medium with no glutamine. Twenty-four hours later, the medium was exchanged again, however this time all flasks received medium containing 4 mM glutamine. The first medium exchange was repeated at 48 hours into the experiment, while the second was repeated at 72 hours into the experiment. Hence the glutamine concentration of the experimental flasks oscillated between 0 mM and 4 mM glutamine over 96 hours, while the glutamine concentration of the control flasks remained constant at 4 mM glutamine. At regular intervals, the flasks were sampled. Total RNA was isolated, medium samples were retained, and isotopic measurements were performed from flasks fed different labelled compounds. All samples taken during the experiment were analyzed. The total RNA harvested was labeled and hybridized to DNA microarrays to measure the abundance of transcripts present. Total RNA control samples were labeled using Cy3 dCTP (Perkin-Elmer), and total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was aliquoted into a 6 uL volume. If concentration of the RNA was necessary, this was conducted using a speed vacufuge (Eppendorf) at 60ºC for 10 minutes. The RNA sample was then mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). This reaction mixture was incubated at 42ºC for approximately two hours. Following the reverse transcription reaction, the RNA template was degraded by addition of 2 uL of 1 N NaOH and incubation at 65 ºC for 10 minutes. The alkaline conditions were then neutralized by addition of 2 uL of 1 N HCl. Samples from identical time points that were to be compared on DNA microarrays were then mixed. The combined sample was cleaned to remove extra primers and nucleotides using a QIAquick Nucleotide Removal kit as described by the manufacturer's instructions. Once cleaned, the sample was concentrated using a speed vacufuge (Eppendorf) for 20 minutes at 65ºC. Following concentration, the sample was resuspended in 32 uL of prewarmed hybridization buffer (Clontech Laboratories), 1 uL was removed for quantitation, and hybridized to prepared oligonucleotide microarrays. Hybridization occurred overnight at 55 ºC using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), and then in 1X SSC, and finally 0.1X SSC. Each wash step was conducted for 20 minutes under high agitation. The arrays were dried by centrifugation and scanned using GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and then analyzed using Matlab (The MathWorks, Inc.). The arrays used for hybridization were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). These arrays contained 17,280 features, printed from a synthesized oligonucleotide mouse library (Operon). Briefly, the library was resuspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed using 32 pins (Telechem), at approximately 50% humidity. Once printed, the probes were crosslinked to the glass slides using a UV Stratalinker (Stratagene). The slides were then blocked for 15 minutes in a 250 mL mixture containing 15.7 g/ L succinic anhydride, 239 mL 1-methyl-2-pyrrolidinone, and 10.7 mL of 1 M boric acid, pH 8.0. They were cleaned by rinsing in 250 mL of milli-Q water (X2), followed by 250 mL of 95% ethanol. They were finally dried using filtered air and stored in the dark at room temperature. Keywords = mouse, liver, hepatoma, cell culture, glutamine, carbon metabolism Keywords: time-course
2003-04-16 | GSE404 | GEO
Project description:A control group consisting of nine C57/BL/6J mice was maintained on a normal diet consisting of Purina Rodent Chow (Harlan Teklad, catalog #5008, 6.5% fat, 49% carbohydrate, 23% protein, 3.5 kcal/ g). The experimental group consisted of nine mice grown on a specialty high-fat diet (Harlan Teklad, catalog #TD88137, 42.16% fat, 42.81% carbohydrate, 15.02% protein, 4.53 kcal/ g). Mice were maintained for ten weeks on their respective diets in a controlled environment with standard light and feeding schedule. Two days before tissue harvest, the experimental group on the high-fat diet was divided into one group of five and one group of four mice. The first group continued on the high-fat diet for the final two days, while the second group fasted. Mouse weights were recorded two days prior, and the day of tissue harvest. At the end of the fasting period liver tissue samples were harvested from each mouse. RNA was purified from the liver tissue samples. Each tissue was homogenized in 1 mL of STAT-60 (Tel-Test, Inc., Friendswood, TX) and the homogenate was mixed with 200 uL of chloroform. The aqueous phase was removed, and mixed with 500 uL of isopropanol to precipitate the RNA. The resulting RNA pellet was washed with 500 uL of 70% ethanol and allowed to air dry. The pellet was dissolved in RNase free water (Ambion, TX), quantified by UV spectroscopy, and stored at -80C. Total RNA control samples (Universal Mouse Reference RNA, catalog # 740100, Stratagene) were labeled using Cy3 dCTP (Perkin-Elmer), and liver total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). The control RNA samples were additionally mixed with 2 uL of random hexamers. The reaction mixtures were incubated at 42 C for two hours. After the reverse transcription reactions, the RNA templates were degraded by addition of 2 uL of 1 N NaOH and incubation at 65 C for 10 minutes. The alkaline conditions were neutralized by addition of 2 uL of 1 N HCl. Liver cDNA samples were then mixed with a control cDNA sample, and the mixtures were cleaned using a QIAquick Nucleotide Removal kit (Qiagen) as described by the manufacturer's instructions. The cleaned samples were concentrated in a speed vacufuge (Eppendorf) for 20 minutes at 65 C, and were dissolved in 32 uL of prewarmed hybridization buffer (Clontech Laboratories), quantified, and hybridized to prepared oligonucleotide microarrays. Hybridization occurred overnight at 55 C using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), then in 1X SSC, and finally in 0.1X SSC. The arrays were dried by centrifugation and scanned using a GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and analyzed using Matlab (The MathWorks, Inc.). Microarrays were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). Arrays contained 16,847 features, printed from a synthesized oligonucleotide mouse library (Operon). The library was suspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed at approximately 50% humidity. Once printed, the probes were crosslinked using a UV Stratalinker (Stratagene). The slides were then blocked for 15 minutes in a 250 mL mixture containing 15.7 g/ L succinic anhydride (Fluka AG), 239 mL 1-methyl-2-pyrrolidinone (EM Science), and 10.7 mL of 1 M boric acid (Fisher), pH 8.0. They were cleaned by rinsing twice in 250 mL of milli-Q water, followed by 250 mL of 95% ethanol (Pharmco Products). After blocking, they were dried using filtered air and stored in the dark at room temperature. In this experiment the gene expression data of the control mice is given by samples GSM6592, GSM6593, GSM6594, and GSM6595. The gene expression data of the high-fat mice is given by samples GSM6599, GSM6600, GSM6601, and GSM6602. The gene expression data of the fasted mice is given by samples GSM6596, GSM6597, and GSM6598. Keywords = mouse, liver, energy homeostasis, gene expression, microarrays Keywords: repeat sample
2003-05-16 | GSE432 | GEO
Project description:Bottom-up mass spectrometric identification of interaction partners of candidate microproteins-GFP fusions after anti-GFP pulldown and tryptic digest
Sample preparation
For each of the conditions, we prepared two biological replicates. For each replicate, a confluent T75 flask of cells was pelleted for five minutes at 300 x g. Pellets were washed with PBS (Sigma-Aldrich, D8537), the suspension centrifuged for five minutes at 300 x g again and the resulting pellet flash frozen in dry ice and Methanol (VWR, 20847.307). Pellets were thawed on ice, resuspended in ice-cold RIPA buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.25 % sodium deoxycholate, 1mM EDTA, 1% NP-40) supplemented with Complete Protease inhibitor (Sigma, 5056489001) and incubated for five minutes on ice. The resulting protein extracts were centrifuged at maximum speed on a table-top centrifuge for 15 minutes at 4C and the supernatant (soluble fraction) incubated with 25 ul GFP-trap magnetic beads (ChromoTek, gtma) for four hours at 4C under constant rotation. The protein-bound beads were then washed once with RIPA buffer, twice with PBS or wash buffer (0.5 % NP-40, 0.1 mM EDTA, 20 mM Tris-HCl pH 7.4, 500 mM NaCl) and once with ddH20. Bound proteins were subsequently eluted twice for five minutes with 15 ul 1% acetic acid. Protein eluates were reduced and alkylated in the same step with 5 mM TCEP (Thermo, PG82080) and 20 mM chloroacetamide (Sigma, C0267) in 250 mM Tris buffer pH 8 for 30 minutes at room temperature. Subsequently, Sera-Mag magnetic bead mix (1:1 ratio of Sigma, GE45152105050250 and GE65152105050250) was added in a 25:1 protein to bead volume ratio and proteins were bound onto the beads by adding an equal volume (protein plus beads) of absolute ethanol. After a five minute incubation, the supernatant was removed and beads were washed three times with 80% ethanol. To perform on-bead digestion, protein-bound beads were resuspended in 50mM TEAB pH 8 (Sigma, T7408) containing 1 ug trypsin (Thermo, 90057) and incubated overnight gently shaking at 37C. Afterwards, beads were allowed to settle on the magnetic rack and the supernatant was taken and kept as the first protein eluate. Subsequently, another elution was performed by adding 50 ul 2% acetonitrile (Sigma, 34851) in 50 mM TEAB. The second elution was combined with the first one and the eluate was dried in a SpeedVac.
2023-05-12 | MSV000091927 | MassIVE