Key result
A protein extraction protocol combining sonication and vortexing with an optimized buffer yielded the highest protein concentration and the most well-resolved protein spots (2163 spots) for 2-DE analysis of chicken bursa of Fabricius.
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An optimized sample preparation protocol combining sonication, vortexing, and a specific extraction buffer significantly improves the quality and yield of 2-DE protein profiles from chicken bursa of Fabricius.
BACKGROUND: Two-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). RESULTS: An 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. CONCLUSION: These 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.
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Wu et al. (2009) studied Healthy and avibirnavirus-infected chickens. Method C (sonication and vortex) with extraction buffer IV vs. Methods A (sonication) and B (vortex), and other extraction buffers was evaluated on Protein yield (mg/g tissue) and number of protein spots on 2-DE gels. A protein extraction protocol combining sonication and vortexing with an optimized buffer yielded the highest protein concentration and the most well-resolved protein spots (2163 spots) for 2-DE analysis of chicken bursa of Fabricius.
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