Dataset Information

M6A binding proteins identidified by RNA pull down.

ABSTRACT: Biotinyted nucleic acid probe assay for protein-m6A interaction

ORGANISM(S): Escherichia Coli

SUBMITTER: Shengce Tao

PROVIDER: PXD037881 | iProX | Wed Nov 02 00:00:00 GMT 2022

REPOSITORIES: iProX

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Publications

Specific pupylation as IDEntity reporter (SPIDER) for the identification of protein-biomolecule interactions.

Jiang He-Wei HW Chen Hong H Zheng Yun-Xiao YX Wang Xue-Ning XN Meng Qingfeng Q Xie Jin J Zhang Jiong J Zhang ChangSheng C Xu Zhao-Wei ZW Chen Zi-Qing ZQ Wang Lei L Kong Wei-Sha WS Zhou Kuan K Ma Ming-Liang ML Zhang Hai-Nan HN Guo Shu-Juan SJ Xue Jun-Biao JB Hou Jing-Li JL Liu Zhe-Yi ZY Niu Wen-Xue WX Wang Fang-Jun FJ Wang Tao T Li Wei W Wang Rui-Na RN Dang Yong-Jun YJ Czajkowsky Daniel M DM Pei JianFeng J Dong Jia-Jia JJ Tao Sheng-Ce SC

Science China. Life sciences 20230414 8

Protein-biomolecule interactions play pivotal roles in almost all biological processes. For a biomolecule of interest, the identification of the interacting protein(s) is essential. For this need, although many assays are available, highly robust and reliable methods are always desired. By combining a substrate-based proximity labeling activity from the pupylation pathway of Mycobacterium tuberculosis and the streptavidin (SA)-biotin system, we developed the Specific Pupylation as IDEntity Repor ...[more]

PMID: 37059927

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Project description:RNA pull-down assay. 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. RNA purification and amplification: 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. Microarray analysis: 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. 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%.

Dataset Information

M6A binding proteins identidified by RNA pull down.

Publications

Specific pupylation as IDEntity reporter (SPIDER) for the identification of protein-biomolecule interactions.

Similar Datasets

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