Identification of linear polyubiquitinated substrates via GST-UBAN pulldown enrichment.
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus Musculus
SUBMITTER: 
Ruihua Xu
PROVIDER: PXD065720 | iProX | Thu Jul 03 00:00:00 GMT+01:00 2025
REPOSITORIES: iProX
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2024-06-22 | PXD048194 | Pride
Project description:GST:Repo-Man(C-terminus) or GST:alone were expressed as in Vagnarelli et al. 2011. Before elution, beads were first incubated with nucleosomes extracted from HeLa. HeLa cells were lysed and nucleosomes were digested with MNase (20min, 37C). Nucleosomes were firstly incubated with GST alone for 2hrs. This cleared lysate was then incubated with GST:Repo-Man and GST alone. Beads were then eluted and mix with 3x Laemli Buffer. Histone bands were sent to Mass Spec.
2016-12-07 | PXD004613 | Pride
Project description:T-47d breast cancer cells were transfected in 6 test/control replicates with biotinylated miR-190b mimic or biotinylated mimic negative control by Qiagen. After 48h cells were lysed and miRNA:mRNA complexes were pulled-down by double precipitation. Firstly by pulling down Ago2 with Dynabeads G with rad Ago2 antibody and secondly via streptavidin coated magnetic beads. The precipitation was carried out according to manufacturer protocol. RNA from the eluted samples was then isolated using phenol-chloroform-isoamyl alcohol mixure and suspended in RNAse free water. This experiment was implemented to assess direct targets bound by miR-190b in the ER-positive breast cancer subtype T-47d.
2022-03-16 | GSE198723 | GEO
Project description:The aim of this project was to determine which viral and host proteins bind to purified recombinant human GTPase Rab11a from lysates of HEK-293T cells infected with influenza A/WSN/33 (H1N1) virus. To this aim we used a GST pull-down assay. Approximately 8∙106 HEK-293T cells were infected with influenza A/WSN/33 (H1N1) virus at a multiplicity of infection of 5 (MOI=5) or mock infected. Twelve hours (h) post-infection cells were harvested by centrifugation at 1,000 rpm for 5 min and lysed for 1 h in lysis buffer [50 mM HEPES-NaOH pH 7.5, 200 mM NaCl, 0.5 % (v/v) IGEPAL CA-630 (Sigma-Aldrich), 1 mM β-mercaptoethanol, 1x PMSF, 1x protease inhibitor (Roche, complete mini, EDTA-free)] at 4 °C. Supernatants were collected by centrifugation at 13,000 rpm for 5 min at 4 °C. Approximately 0.2 mg of purified GST-tagged Rab11CA (constitutively active Rab11a with a Q70L substitution) or GST tag was bound to pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) (100 µl slurry per 1 ml sample volume) at 4 °C for 3 h in binding buffer [50 mM HEPES-NaOH pH 7.5, 300 mM NaCl, 10 % (v/v) glycerol, 8 mM MgCl2, 10 µM GTP-γ-S (Abcam)]. GST-Rab11CA or GST tag bound beads were incubated with virus infected or mock infected cell lysates for 1 h at 4 °C followed by five washes in binding buffer. Rab11CA and bound proteins were released in the same buffer supplemented with 1 mM DTT and 0.1 mg human rhinovirus (HRV) 3C protease to cleave off Rab11CA from the GST tag. After 1 h incubation at 4 °C released proteins were cleared from the beads by centrifugation at 1,000 rpm for 5 min at 4 °C. GST pull-down protein fractions were used for analysis by mass spectrometry.
2022-01-17 | PXD030058 | Pride
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
Project description:The chromatin fraction was isolated from 5x107 HeLa cells. Chromatin was digested in 100 mL of MNase (40 units/ mL ) reaction buffer for 3 min at 37 °C in a thermomixer (1,400 rpm). After addition of 10 L EGTA (25mM) to inactivate MNase, soluble digested chromatin was collected by 13,000 rpm centrifuge for 5 min. 10 mg of Pol II antibody was conjugated to magnetic protein A beads. The supernatant was diluted with 400 mL of NET-2 buffer and pSer2-Pol II antibody-conjugated beads were added. Incubated the IP reaction at 4 °C for 1 hr. The beads were washed with 1 mL of NET-2 buffer six times and 100 mL of 1xPNKT (1xPNK buffer and 0.05% Triton X-100) buffer once. Washed beads were incubated in 50 mL PNK reaction mix (1xPNKT, 1 mM ATP and 0.05 U/ml T4 PNK (NEB)) in Thermomixer at 37 °C, 1,400 rpm for 6 min. Beads were washed with 1 mL of NET-2 buffer once and RNA was extracted with Trizol reagent. RNA was suspended in urea Dye (7M Urea, 1xTBE, 0.1% BPB and 0.1% XC) and resolved on 6% TBU gel (Invitrogen) at 200 V for 5 min. Gels with 30-160 nt RNAs were collected. 0.2 mL tube was prepared with holes made with 25G needle and placed in a 1.5 mL tube. Gel fragments were placed in the layered tube and broken down by centrifugation at 12,000 rpm for 1 min. The small RNAs were eluted from gel using RNA elution buffer (1 M NaOAc and 1 mM EDTA) at 25 °C for 1 hr in Thermomixer (900 rpm). Eluted RNA was purified with SpinX column (Coster) with 2 glass filters (Millipore) and the flow-through RNA was ethanol precipitated.
2019-10-15 | GSE134102 | GEO
Project description:U2OS osteosarcoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) and Raji B-cells in RPMI 1640 medium (Gibco) supplemented with 10% fetal calf serum (FCS, Bio&Sell), 2 mM L-glutamine (Gibco), 100 U/mL penicillin (Gibco), and 100 µg/mL streptomycin (Gibco) at 37°C at 8% or 5% CO2, respectively. Chromatin immunoprecipitation for ChIP-seq: Cells were crosslinked using a formaldehyde containing solution (10mM NaCl, 0,1mM EDTA pH 8, 0,05mM EGTA pH 8, 5mM HEPES pH 7.8 and 1% formaldehyde) for 10 minutes at 20°C, the reaction was quenched by the addition of glycin to a final concentration of 250uM for 5 minutes. Crosslinked cells were collected and washed twice with PBS before snap freezing in liquid nitrogen and storage at -80°C until subsequent use. Prior to sonication, the crosslinked cells were resuspended in lysis buffer (50mM Hepes pH 7.5, 140mM NaCl, 1mM EDTA pH 8, 10% glycerol, 0.75% NP-40, 0.25% Triton X-100, 1X protease inhibitor cocktail) at 4°C for 20 minutes. Nuclei were collected by centrifugation and washed in a second buffer (200mM NaCl, 1mM EDTA pH 8, 0.5mM EGTA pH 8, 10mM Tris pH 8, 1X protease inhibitor cocktail) for 10 minutes at 4°C then collected by centrifugation and resuspended in the shearing buffer (1mM EDTA pH 8, 0.5mM EGTA pH 8, 10mM Tris pH 8, 100mM NaCl, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, 1X protease inhibitor cocktail). Sonication was carried out in a Bioruptor Pico ultrasounds water bath (Diagenode B01060001) for 30 cycles of 30 sec ON and 30 sec OFF pulses in 4°C water. Sonicated extracts were centrifuged at high speed in the presence of 0,1% of Triton X-100 and snap frozen in liquid nitrogen then stored at -80°C until subsequent use. Prior to ChIP, the ZNF768 mAb was coupled to protein-G coated magnetic beads (Dynabeads, life technologies) by incubation in 0,5% BSA PBS overnight at 4°C. Pre-coated beads were then washed and incubated with the sonicated chromatin extracts, the ChIP was carried out overnight at 4°C on a rotating wheel. The equivalent of 10 × 107 cells sonicated extract was used for each ZNF ChIP experiment for both cell lines. After incubation, the beads were washed 7 times with Wash buffer (50mM Hepes pH 7.6, 500mM LiCl, 1mM EDTA pH 8, 1% NP-40, 0.7% Na-Deoxycholate, 1X protease inhibitor cocktail) followed by one wash with TE-NaCl buffer (10mM Tris pH 8 and 1mM EDTA pH 8, 50mM NaCl). Immunoprecipitated chromatine was eluted by two sequential incubations with 100µl Elution buffer (50mM Tris pH 8, 10mM EDTA pH 8, 1% SDS) at 65°C for 15 minutes. The two eluates were pooled and incubated at 65°C for 12 hours to reverse-crosslink the chromatin followed by treatment with RNase A (0.2µg/mL) at 37°C for two hours and Proteinase K (0.2µg/mL) at 55°C for two hours. The DNA was isolated by phenol:chloroform: isoamylalcohol (25:24:1 pH 8) extranction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA). At least 1ng of ChIP DNA was used to prepare sequencing library with Illumina ChIP Sample Library Prep Kit (Illumina, USA) with a few optimizations to the protocol. The ChIP DNA was size selected using Ampure beads (Life technologies) to enrich for fragments < 400Bp prior to end-repair, 3’end adenylation and adapter ligation. Library fragments were then directly amplified by 10 cycles of PCR.
2018-10-29 | GSE111879 | GEO
Project description:pGEX-4T-1-B2M was constructed using the GST gene fusion system according to the manufacturer’s instructions. To produce glutathione S-transferase (GST) and GST-B2M fusion proteins, pGEX-4T-1 and pGEX-4T-1-B2M were individually transfected into BL21 cells. Protein expression was induced by ispropylb-D-1-thiogalactopyranoside (IPTG). GST-B2M and GST proteins were purified by binding to Glutathione Sepharose-4B beads. Then, beads were incubated with mouse brain lysates from C57BL/6J mice at 4 °C for 12 h on a rotator, and then were washed with the washing buffer three times. Beads-bound complexes were eluted by boiling, and subjected to SDS-PAGE and silver staining. The proteins were identified by Mass Spectrometry.
2023-01-04 | PXD039274 |
Project description:pGEX-4T-1-B2M was constructed using the GST gene fusion system according to the manufacturer’s instructions. To produce glutathione S-transferase (GST) and GST-B2M fusion proteins, pGEX-4T-1 and pGEX-4T-1-B2M were individually transfected into BL21 cells. Protein expression was induced by ispropylb-D-1-thiogalactopyranoside (IPTG). GST-B2M and GST proteins were purified by binding to Glutathione Sepharose-4B beads. Then, beads were incubated with mouse synaptosome fractions from C57BL/6J mice at 4 °C for 12 h on a rotator, and then were washed with the washing buffer three times. Beads-bound complexes were eluted by boiling, and subjected to SDS-PAGE and silver staining.
2023-01-04 | PXD039275 |
Project description:Glioblastoma stem cells (TS-543) were harvested and cross-linked with 1% formaldehyde for 10 mins at room temperature. The cells were lysed using SDS Lysis buffer for ChIP (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). The lysate was then sonicated for 25 cycles at 30% amplitude (15 s ON and 45 s OFF). The sonicated samples were then diluted in ChIP dilution buffer (0.01% SDS, 1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8, 167 mM NaCl) and used for the immunoprecipitation with H3K27ac (Abcam, Ab4729), H2AZ (Active motive, cat#39113) and H3K27ac (Abcam cat#ab8580). After an over-night incubation with antibody, the bound DNA was washed sequentially with low salt wash buffer (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP40, 1%deoxycholate, 1 mM EDTA, 10 mM Tris–HCl pH 8) and TE wash buffer (10 mM Tris–HCl pH 8, 1 mM EDTA) to remove non-specific sequences and eluted in the elution buffer (84 mg NaHCO3, 1 ml 10% SDS, 9 ml H2O). Then the samples were reverse cross-linked using NaCl at 65°C overnight. The eluted DNA was purified and used for library preparation. Library preparation was performed as previously described as described in Bowman et al. BMC Genomics 2013. Briefly, end repair with NEBNext End Repair enzyme (NEB, E6050) and clean-up with 2.4x volume AMPure XP beads (Beckman Coulter, A63880). A-tailing with Klenow Fragment (NEB, M0212) and purified with 2.4x volume AMPure XP beads. Adapter ligation reactions contained annealed universal adapter and T4 rapid ligase (NEB, B0202) and clean-up with 1.8x volume AMPure XP beads. Chromatin was amplified using KAPA Real-time Library Amplification Kit (KAPABIOSYSTEMS, KK2701) with universal primer and barcoded primer. Amplified chromatin was purified with QIAquick Gel Extraction Kit (Qiagen) and sequenced paired-end with Illumina NextSeq 500.
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