Screening for proteins interacted with IDH1 in PC-9 cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Fang Hua
PROVIDER: PXD046560 | iProX | Wed Nov 01 00:00:00 GMT 2023
REPOSITORIES: iProX
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Project description:Proteins in PC-9 cells were extracted by CO-IP lysis buffer, PHGDH antibody and agarose gel beads were added into the lysis buffer and rotated in 4°C overnight. The agarose gel beads were washed for 5 times by centrifugation, and denatured in 2 x loading buffer. The denatured protein samples were sent for MS analysis and screened for the proteins interacted with PHGDH.
2023-11-01 | PXD046559 | iProX
Project description:Proteins in RAW264.7 cells were extracted by CO-IP lysis buffer, Id1 antibody and agarose gel beads were added into the lysis buffer and rotated in 4°C overnight. The agarose gel beads were washed for 5 times by centrifugation, and denatured in 2 ✖️loading buffer. The denatured protein samples were sent for MS analysis and screened for the transcriptional factors interacted with Id1.
2023-10-17 | PXD046173 | iProX
Project description:The coiled-coil domain of EPG5 was synthesized as follows: Biotin-MNKNEAVSQQIHVLQKEVRQLQAE AAQ (from Beijing SciLight Biotechnology, LCC). 1 mg of this short peptide was incubated with ES cells lysates followed by immunoprecipitation with anti-biotin beads. The precipitates were extensively washed with lysis buffer then boiled with SDS loading buffer and subjected to SDS–PAGE. Total proteins were excised from the gels, then subjected to in-gel digestion and analysis by LC-MS/MS.
2019-02-25 | PXD012563 | Pride
Project description:The CLIP procedures were conducted using HeLa cells according to the previous study (Liu et al., 2016) with slight modifications. Briefly, 4 plates of HeLa cells in 15 cm dishes were transfected with pPB/ALKBH3 plasmid for 48 h to reach about 90% cell confluency. After washing twice with ice-cold PBS, cells were irradiated twice with 400 mJ/cm2 at 254 nm by Stratalinker on ice. Cells were lysed in high salt lysis buffer (300 mM NaCl, 0.2% NP-40, 20 mM Tris-HCl PH 7.6, 0.5 mM DTT, protease inhibitor cocktail (1 tablet/50 ml), and RNase inhibitor (1:200)) at 4°C for 30 min. Supernatant was collected after centrifuged at 17,000 g at 4°C for 15 min and further treated with 1 U RNase T1 for 15 min at 24 °C. After centrifugation and filtration through 0.22 µM filter, 10% supernatant was collected as input for further sequencing analysis. Anti-Flag M2 beads (Sigma-Aldrich, St. Louis, MO, 80 μl) were washed three times with lysis buffer and incubated with the filtered supernatant at 4°C for 4 hrs. After removing the flow through by magnetic rack, beads and input were further incubated with 10 U RNase T1 exactly for 8 min. Samples were treated with 95 µL commercial PNK buffer and 0.5 U PNK for 15 min at 37 °C. After treatments, final concentration of 100 µM ATP and 1 U PNK were added and incubated at 37 °C for another 30 min. The RNA/protein complexes were eluted by NuPAGE 1 × loading buffer and fractionated by neutral NuPAGE 4–12% bis-tris gel. After cutting the gel, RNAs were recovered by proteinase K digestion in proteinase K buffer, followed by phenol/chloroform extraction using glycogen as carrier. The RNA fragments were ligated to adapter and established library by NEB small RNA library preparation kit (E7330S).
2022-03-01 | GSE112625 | GEO
Project description:To analyze MARVELD1 interacting proteins, HeLa cells were transfected with MARVELD1-Flag or pcDNA3.1 (PC), then treated with 8 mM Hydroxyurea (MCE, USA) or DMSO for 24 h, and subjected to IP assays with anti-DYKDDDDK magnetic agarose (Thermo, USA). After washing five times with PBS buffer, samples were boiled in 2× SDS loading buffer, resolved in SDS-PAGE, visualized by Coomassie Blue staining, and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis (PTM Bio, China, project number: FA203GI).
2023-05-10 | PXD034445 | Pride
Project description:Purpose: The goal of this study is to compare DAXX genome binding/occupancy by high throughput ChIP sequencing in control, wild type DAXX overexpressd cells (WT DAXX OE), and wild type DAXX SUMO interacting motif mutant overexpressd cells (DSM OE). Methods: The panel of MDA_MB-231 derived cell lines (control, WT DAXX OE, DSM OE) were cultured in complete DMEM. At about 90% confluency, the cells were crosslinked by adding 37 % formaldehyde to the final concentration of 1% for 10 min at room temperature. Crosslinking was stopped by adding glycine to the final concentration of 125 mM. Cells were lifted, washed with cold PBS, and pelleted by centrifugation. The cells were resuspended in a swelling buffer in the presence of the protease inhibitor cocktail (Sigma) and then pelleted and resuspended in the SDS lysis buffer. The lysates were transferred to a Covaris microTUBE and sonicated with an E220 Covaris Ultrasonicator. Chromatin fragmentation (~500 bps) was verified through agarose gel electrophoresis. The fragmented chromatins were diluted and incubated with a control IgG and the DAXX mAb (5G11) along with protein A/G magnetic beads. The beads were washed sequentially with a low salt buffer, high salt buffer, LiCl buffer, and TE buffer (twice). The immunoprecipitated chromatins were eluted at 65 °C for 15 min, and the eluted chromatins were subjected to proteinase K digestion at 65 °C for 3 h. The DNAs were recovered through a Qiagen mini-prep column. The immunoprecipitated DNAs were used for qPCR and library construction and high throughput sequencing using an Illumina Hi-Seq 2500 sequencer. Results: We surveyed genome-wide occupancy of DAXX using ChIP-seq technology. Overexpression of WT DAXX increased DAXX?s chromatin association specifically with lipogenic pathway genes where DSM OE reverse. Conclusions: Our study represents the first detailed analysis of DAXX occupancy in lipoegenic gene transciption.
2021-12-20 | GSE190783 | GEO
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:PTB is multifunctional RNA binding protein reported to be involved in splicing, 3' -end processing, stability and translational regulation. Sample preparation methods (Gama-Carvalho et al, 2006, submitted) Suspension HeLa cells were grown in DMEM, 10% FCS, Pen/Strep and split 1:2 the day before harvesting. Post-nuclear cytoplasmic extracts were obtained as described (Herbert and Hecht 1999), using 5x107 cells/ml of lysis buffer. mRNAs were isolated by immunoprecipitation from 200ul of precleared HeLa cell extract, using the anti-U2AF65 MC3 mAb (Gama-Carvalho, Krauss et al. 1997) or the Bb7 anti-PTB mAb (ATCC) for 2h at 4ºC. Immune complexes were precipitated with a slurry with 50% of protein A/protein G agarose beads (Amersham), blocked with 100ug/ul of tRNA and RNAse free BSA (Ambion), by rotating for 1h at 4ºC and washed with lysis buffer. Complexes bound to the beads were eluted with TES buffer (10mM Tris, 0.5M EDTA, 0.5% SDS, pH 8.0) by heating at 65ºC for 10 minutes and Trizol extracted. Polyadenilated RNA in the cell extract (input) and in the immunoprecipitation sample was reverse transcribed, end-tagged and PCR amplified using the Super SMART PCR cDNA synthesis kit from Clontech, following manufacturer instructions. 1ug of input or precipitated RNA was used per RT-PCR reaction to produce samples for microarray hybridization. For each set of samples (input and immunoprecipitated) a 100ul test PCR reaction was performed according to manufacturer instructions and analyzed by agarose gel electrophoresis to select ideal conditions for amplification in the linear range. PCR reactions were pooled to obtain enough material for microarray hybridization. 15ug of PCR amplified cDNA from input and immunoprecipitated samples were fragmented, end labeled with biotin and hybridized to Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays as described (Brodsky, Meyer et al. 2005), following standard Affymetrix hybridization protocols. Brodsky, A. S., C. A. Meyer, et al. (2005). "Genomic mapping of RNA polymerase II reveals sites of co-transcriptional regulation in human cells." Genome Biol 6(8): R64. Gama-Carvalho, M., R. D. Krauss, et al. (1997). "Targeting of U2AF65 to sites of active splicing in the nucleus." J Cell Biol 137(5): 975-87. Herbert, T. P. and N. B. Hecht (1999). "The mouse Y-box protein, MSY2, is associated with a kinase on non-polysomal mouse testicular mRNAs." Nucleic Acids Res 27(7): 1747-53. Keywords: RNA immunoprecipitation
2006-10-14 | GSE6021 | GEO
Project description:Immunoaffinity Purifications of Human Argonaute Twelve hours after transfections with mock or with miR-124, we washed each 15-cm plate once with phosphate-buffered saline (usually two plates were used per IP), then added 1 ml of 4 C lysis buffer (150 mM KCl, 25 mM Tris-HCl [pH 7.4], 5 mM Na-EDTA [pH 8.0], 0.5% Nonidet P-40, 0.5 mM DTT, 10 ul protease inhibitor cocktail [Pierce Cat# 78437], 100 U/ml SUPERaseIn [Ambion Cat# AM2694]). Following a 30-min incubation at 4 C, we scraped the plates, combined the lysates, and then spun them at 4 C for 30 min at 14,000 RPM in a microcentrifuge. We collected the supernatant and filtered it through a 0.45-um syringe filter. We froze an aliquot of lysate in liquid nitrogen for reference RNA isolation. We then added 0.22 mg/ml heparin to the lysate. We mixed the lysate with 2.5 mg of Dynal m-280 streptavidin beads (250 ul from original storage solution) coupled to biotinylated 4F9 ago antibody (~12.5 ug), which we equilibrated immediately prior to use by washing twice with 1 ml of lysis buffer. We incubated the beads with the lysate for 2 h at 4 C and then washed the beads twice with 1.25 ml of ice-cold lysis buffer for 5-min each. Five percent of the beads were frozen for SDS PAGE analysis after the second wash. RNA was extracted directly from the remaining beads using lysis buffer from Invitrogen's Micro-to-Midi kit (Invitrogen Cat# 12183-018). We purified RNA from the lysate and RNA extracted from the beads with the Micro-to-Midi kit as per vender's instructions, except that the percentage isopropanol used for binding to the column was 70%, instead of 33%, to promote the binding of small RNAs. RNA was amplified with ambion kit 1755. RNA from total cell lysate was labeled with cy3 and IPd RNA was labeled with cy5
2009-11-10 | GSE18834 | GEO
Project description:For identification of Mi-2β-interacting proteins, B16F10 cells (1 × 108) were collected and washed three times with PBS and lysed in lysis buffer on ice for 30 min. Cell lysates were collected and then isolated over night by anti-Mi-2β magnetic agarose beads. The anti-Mi-2β magnetic agarose beads were collected and washed three times with PBS. The prepared protein samples were incubated with sample buffer for 15 min at 100 °C, and then separated by SDS-PAGE (10%). The gel was immersed in staining solution (0.3% Coomassie blue, 45% methanol, 10% glacial acetic acid and 45% dH2O) on shaker for 30 min, followed by incubation in destaining solution (20% methanol, 10% glacial acetic acid, and 70% dH2O) on the shaker overnight. The bands were excised and sent to Biological Mass Spectrometry Facility of Shanghai Applied Protein Technology Co., Ltd for protein identification.
2024-02-18 | PXD049467 | iProX