RNA-蛋白质印记法研究水稻幼穗中蛋白质-RNA的相互作用

Saminathan Thangasamy; Guang-Yuh Jauh

doi:10.21769/BioProtoc.625

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Peer-reviewed

Northwestern Blot of Protein-RNA Interaction from Young Rice Panicles

RNA-蛋白质印记法研究水稻幼穗中蛋白质-RNA的相互作用

Saminathan Thangasamy email

Guang-Yuh Jauh email

发布: 2013年04月05日第3卷第7期 DOI: 10.21769/BioProtoc.625 浏览次数: 15882

评审: Tie Liu

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The authors used this protocol in:

Cover of The Plant Journal, featuring study using the protocol.

Jul 2012

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Abstract

The northwestern assay is employed to study the interaction between protein and RNA. The RNA binding proteins tend to bind to different kinds of RNA through either known domains or unknown sequences of proteins. Rice LGD1 recombinant protein, a grass-specific novel protein with RNA binding sequences in its C-terminal, was used to probe its function as an RNA binding protein. The LGD1 comprising von Willebrand factor type-A domain (vWA), coiled-coil and nuclear localization signal (NLS) is a class of protein that localizes both in the nucleus and cytoplasm. Although LGD1 does not contain any putative RNA binding domains, we could find high-affinity RNA binding residues at the C-terminus using ‘RNABindR’ prediction software (Terribilini et al., 2007). The LGD1 recombinant protein, purified from bacteria, somehow forms both dimer and monomer even under denaturing conditions. However, only the dimeric form is able to bind to total and mRNAs. Due to its reproducibility and reliability, we believe that this protocol can be used across different organisms.

Keywords: LGD1 (lgd1)

Multiple transcripts (多个转录)

RNA binding (RNA结合)

Alternative splicing (选择性剪接)

Rice (水稻)

Materials and Reagents

Young panicle tissues of wild type (Oryza sativa L. ssp. Japonica cultivar Tainung67) plants
Tissues were collected from young panicles while they were in secondary branch initiation
Recombinant GST-LGD1 protein from E. coli (BL21)
pGEX-4T-1 GST expression vector (GE Healthcare, catalog number: 28-9545-49 )
Total RNA isolation kit - Trizol (Life Technologies, Invitrogen^TM, catalog number: 15596-026 )
mRNA purification kit - Oligotex mRNA Mini Kit (QIAGEN, catalog number: 72022 )
RNA 5'-end Labeling kit (T4 Polynucleotide Kinase) (Thermo Fisher Scientific, catalog number: EK0031 )
FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific, catalog number: EF0651 )
[γ-³²P]-ATP–for isotope labeling
PVDF Immobilon-P membrane (EMD Millipore, catalog number: IPVH15150 )
G-50 gel filtration dye terminator removal column (Geneaid, Taiwan, catalog number: CG050 )
Primary anti-GST antibody (Bioman, Taiwan, catalog number: GST001M ); HRP-conjugated secondary antibody (Thermo Fisher Scientific, catalog number: SA1-9510 )
SuperSignal West Pico Chemiluminescence detection system (Thermo Fisher Scientific, catalog number: 34087 )
Blocking buffer (see Recipes)

Equipment

Gel electrophoresis (Bio-Rad Laboratories, catalog number: 165-8005EDU )
SNAP i.d.^® 2.0 protein detection system
Equipment for phosphor imaging (Bio-Rad Laboratories, catalog number: 170-9460 )

Procedure

English

中文翻译

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如何引用

Thangasamy, S. and Jauh, G. (2013). Northwestern Blot of Protein-RNA Interaction from Young Rice Panicles. Bio-protocol 3(7): e625. DOI: 10.21769/BioProtoc.625.