Optimized sample preparation for two-dimensional gel electrophoresis of soluble proteins from chicken bursa of Fabricius

Yongping Wu,Xuetao Jiang,Jiyong Zhou,Junhua Wang,Xin Zhang,Lixue Shi,Xiaojuan Zheng,Wei Yin

doi:10.1186/1477-5956-7-38

Yongping Wu, Xuetao Jiang + Show 6 more

Open Access

https://doi.org/10.1186/1477-5956-7-38

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Abstract

BackgroundTwo-dimensional gel electrophoresis (2-DE) is a powerful method to study protein expression and function in living organisms and diseases. This technique, however, has not been applied to avian bursa of Fabricius (BF), a central immune organ. Here, optimized 2-DE sample preparation methodologies were constructed for the chicken BF tissue. Using the optimized protocol, we performed further 2-DE analysis on a soluble protein extract from the BF of chickens infected with virulent avibirnavirus. To demonstrate the quality of the extracted proteins, several differentially expressed protein spots selected were cut from 2-DE gels and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).ResultsAn extraction buffer containing 7 M urea, 2 M thiourea, 2% (w/v) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 50 mM dithiothreitol (DTT), 0.2% Bio-Lyte 3/10, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 U/ml Deoxyribonuclease I (DNase I), and 0.25 mg/ml Ribonuclease A (RNase A), combined with sonication and vortex, yielded the best 2-DE data. Relative to non-frozen immobilized pH gradient (IPG) strips, frozen IPG strips did not result in significant changes in the 2-DE patterns after isoelectric focusing (IEF). When the optimized protocol was used to analyze the spleen and thymus, as well as avibirnavirus-infected bursa, high quality 2-DE protein expression profiles were obtained. 2-DE maps of BF of chickens infected with virulent avibirnavirus were visibly different and many differentially expressed proteins were found.ConclusionThese results showed that method C, in concert extraction buffer IV, was the most favorable for preparing samples for IEF and subsequent protein separation and yielded the best quality 2-DE patterns. The optimized protocol is a useful sample preparation method for comparative proteomics analysis of chicken BF tissues.

Highlights

Two-dimensional gel electrophoresis (2-DE) is a powerful method to study protein expression and function in living organisms and diseases
Optimization of protein extraction methods Sample preparation prior to isoelectric focusing (IEF) is an important step for separation of proteins from a complex sample in two-dimensional gel electrophoresis (2-DE)
Using chicken bursa of Fabricius (BF) tissues, which contain many contaminants that strongly interfere with 2-DE, resulting in streaking and smearing, we optimized a protocol for protein extraction and 2-DE

Summary

Introduction

Two-dimensional gel electrophoresis (2-DE) is a powerful method to study protein expression and function in living organisms and diseases. This technique, has not been applied to avian bursa of Fabricius (BF), a central immune organ. Substantial efforts have been devoted to optimizing the preparation and handling of biological samples in order to enhance the quality of two-dimensional (2-D) gels [22,23,24,25,26,27,28], but since the diversity of tissue organization and protein content affect protein solubility, sample preparation must be optimized on a case-by-case basis [29]

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Discussion

Conclusion