RNA pull-down assay
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus Musculus (mouse)
SUBMITTER: Satoshi Yokoi
PROVIDER: PXD007521 | JPOST Repository | Tue Aug 21 00:00:00 BST 2018
REPOSITORIES: jPOST
Dataset's files
Items per page: 5 1 - 5 of 18 |
Similar Datasets
Project description:For the GST pull-down of DRG ganglion, the GST- or Tmc7-GST-associated beads were further incubated with cell lysates derived from DRG ganglion. The pulled-down proteins were then separated by SDS-PAGE gel electrophoresis and subsequently stained with silver staining Kit. Finally, the target molecular weight bands and undenatured beads were analyzed by proteomic analysis, respectively.
2024-03-08 | PXD050466 |
Project description:ZFP36L1 is a tandem zinc-finger RNA-binding protein that recognizes conserved Adenylate-Uridylate-rich Elements (AREs) located in 3' untranslated regions (UTRs) to mediate RNA decay. We hypothesized that ZFP36L1 is a negative regulator of a post-transcriptional hub involved in the RNA half-life regulation of cancer-related transcripts. Forced expression of ZFP36L1 in cancer cells markedly reduced cell proliferation in vitro and in vivo; whereas silencing of ZFP36L1 enhanced tumor cell growth. To identify direct downstream targets of ZFP36L1, systematic screening using RNA pull-down of wildtype and mutant ZFP36L1 as well as whole transcriptome sequencing of bladder cancer cells ± tet-on ZFP36L1, was performed. A network of 1,410 genes was identified as potential direct targets of ZFP36L1, including HIF1A, CCND1, and E2F1. ZFP36L1 specifically bound to the 3' UTRs of these targets for RNA degradation, thus suppressing their expression. Collectively, our findings reveal an indispensable role of ZFP36L1 as a post-transcriptional safeguard against aberrant hypoxic signaling and abnormal cell cycle progression.
2020-10-02 | GSE136179 | GEO
Project description:Long non-coding RNAs (lncRNAs) work together with diverse RNA-binding proteins (RBPs) to fulfill key regulations in important cellular functions. Here, we present a protocol to detect lncRNA-RBP interactions in vitro using a tRNA scaffold containing a streptavidin aptamer pull-down assay. We describe steps for preparing both protein and lncRNA transcripts, lncRNA-protein interaction detection with an in vitro binding assay, and western blot analysis. This protocol is applicable to screen for RNA-interacting proteins using cell lysates followed by mass spectrometry analysis. For complete details on the use and execution of this protocol, please refer to Yang et al. (2023).1.
| S-EPMC10789616 | biostudies-literature
Project description:We aimed to discover trans-acting RNA molecules involved in mRNA 3’ processing. We reasoned that, if there exist such functional RNAs, they must directly associate with the key machinery responsible for mRNA 3’ processing. Therefore, it would be of great value to comprehensively identify RNAs interacting with pre-mRNA 3’ processing complex. To this goal, we took advantage of previously well-characterized system combined with high-throughput sequencing to investigate the target RNAs at the transcriptomic level. Briefly, we used polyA site (PAS) RNA substrates, SV40 late (SVL), and corresponding control RNAs with point mutation (U to C) at the highly conserved cis-element AAUAAA. RNA substrates were first biotinylated at the 3’ end, and then bound to the streptavidin magnetic beads. After incubation with Hela Nuclear Extract (NE) under polyadenylation condition, the two wild type RNA substrates could specifically recruit mRNA 3’ processing factors in NE for complex assembly. The purification of the protein complex and its interacting RNAs were performed using biotin-streptavidin pull-down. We extracted RNAs from the pull-down sample, prepared the strand-specific RNA-Seq libraries and submitted them for deep-sequencing.
2017-07-01 | GSE98218 | GEO
Project description:RNA pull-down assay.<br>For the recombinant protein pull-down assays, 50 M-5g of recombinant His-tag TcRBP40 protein were bound to 100 uL of Ni-NTA resin (Qiagen) overnight at 4M-0C. 100 M-5g of total RNA from epimastigotes were incubated with the bound protein in 500 M-5l EMSA buffer at 4M-0C for 2 h, in the presence of Heparine and Spermidine as competitors. Bounded and supernatant samples were separated. The bound sample was washed with the same buffer three times, soft-mixing for 10 min each. After washing, RNA present in the bound and supernatantM- fractions were purified.<br><br>RNA purification and amplification:<br><br>RNA was extracted using the RNeasy mini kit (Qiagen). Linearly amplified RNA (aRNA) was generated with the MessageAmpM-^YII aRNA Amplification kit (Ambion), according to the manufacturerM-^Rs manual.<br><br>Microarray analysis:<br>The microarray was constructed with 70-mer oligonucleotides. Due to the hybrid and repetitive nature of the sequenced T. cruzi strain, all coding regions (CDS) identified in the genome (version 3) were retrieved and clustered by the BLASTClust program, using parameters of 40% coverage and 75% identity. For probe design, it was used ArrayOligoSelector software (v. 3.8.1), with a parameter of 50% G+C content. Was obtained 10,359 probes for the longest T. cruzi CDS of each cluster, 393 probes corresponded to the genes of an external group (Cryptosporidium hominis) and 64 spots contained only spotting solution (SSC 3x), given 10,816 spots in total. These oligonucleotides were spotted from a 50 M-5M solution onto poly-L-lysine coated slides and cross-linked with 600 mJ UV. Each probe corresponding to the T. cruzi genes was identified according to the T. cruzi Genome Consortium annotation (www.genedb.org). We compared bound and unbound mRNA, extracted from two independent pull-down assays, in a dye-swap design including four slides. <br>Microarray images were analyzed by Spot software (Spot). The Limma package (Smyth GK, 2004) was used for background correction by the normexp method, intra-slide normalization by the printtiploess method and inter-slide normalization by the quantile method. The results for the two intra-slide probe replicates were then averaged. The pull-down results were averaged, and probes displaying more than a two-fold difference between the bound and unbound fractions were selected, at FDR 1%.
2012-06-30 | E-MEXP-3057 | biostudies-arrayexpress
Project description:To capture endogenous DANCR transcripts, we designed and synthesized 11 5′ biotinylated 90-mer DNA oligonucleotides antisense that spanned the entire length of the target RNA DANCR. Pull-down proteins were separated by SDS-PAGE gel and stained with Fast Silver Stain Kit (Beyotime, China). Cell lysates were incubated with biotinylated DNA probes (10 pmol) and 50 μL of streptavidin-agarose beads (Invitrogen) for 1 hour. The proteins were precipitated and diluted in 50 μl of digestion buffer (8 M urea, 50 mM Tris-HCl pH 7.8), separated by SDS-PAGE gel electrophoresis and visualized by silver staining using the PAGE Gel Silver Staining Kit (Solarbio, Beijing, China) as the protocol described. Specific bands were excised for proteomics screening by mass spectrometry analysis (Tianjin Novogene Bioinformatic Technology, Tianjin, China).
2022-11-15 | PXD038169 |
Project description:To identify the protein partners of SARS-CoV-2 -1 PRF RNA, we performed RNA pull-down assays using streptavidin magnetic beads. To obtain more accurate and reliable data, tRSA sequence-tagged -1 PRF and -1 FSE RNA probes were both used to capture binding proteins in our screen. As a random sequence control for nonspecificity, the equivalent proteins extracted from H1299 cells were incubated with tRSA-labeled -1 PRF and -1 FSE RNA probes. A high-throughput technology, LC/MS-MS(MS), was applied to identify the candidate interacting proteins of SARS-CoV-2 -1 PRF RNA.
2023-05-25 | PXD042504 |
Project description:RNA pull down assay of GST-ZFP36L1 (wildtype and mutant)
| PRJNA561512 | ENA
Project description:RNA pull-down was used to identified the interacting protein of circularRNA-FGL1
2024-04-18 | PXD051546 |
Project description:Biotinyted nucleic acid probe assay for protein-m6A interaction
2022-11-02 | PXD037881 |