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Characterization of Plant Polyadenylation Complexes by Using Tandem Affinity Purification

  • Hongwei ZhaoEmail author
  • Xinfu Ye
  • Qingshun Quinn Li
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1255)

Abstract

Messenger RNA in eukaryotic cells is initially produced as a nascent transcript (pre-mRNA) without a polyadenine [poly(A)] tail to the 3′ end. The precise cleavage of the pre-mRNA and addition of a poly(A) track need the communication between cis-elements in the pre-mRNA sequences and transacting protein factors recognizing them. Based on homology analyses, Arabidopsis cleavage and polyadenylation specificity factor (AtCPSF) complex should play a critical role in pre-mRNA 3′ end processing. Here we describe the isolation of AtCPSF complex by using a tandem affinity purification (TAP) method. We demonstrate that TAP is a potent protein complex isolating approach that can fulfill a downstream protein identification purpose based on mass spectrometry techniques.

Key words

Pre-mRNA Polyadenylation factor Tandem affinity purification Calmodulin binding protein TEV Protein purification Mass spectrometry 

Notes

Acknowledgement

We received funding support from the US National Science Foundation (grant nos. IOS–0817829 and IOS-1353354 to QQL). QQL received funding support from the Fujian Hundred Talent Plan.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Plant Pathology, College of Plant ProtectionNanjing Agricultural UniversityNanjingChina
  2. 2.Institute of Fruit TreesFujian Academy of Agricultural ScienceFuzhouChina
  3. 3.Rice Research InstituteFujian Academy of Agricultural ScienceFuzhouChina
  4. 4.Department of BiologyMiami UniversityOxfordUSA

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