ÃÞ »¨ ѧ ±¨ Cotton Science 2009£¬21(1)£º3-9
Protein Extraction from Embryogenic Cultures of Cotton for Two-dimensional Gel Electrophoresis Abstract: A new preparation protocol of protein extracts was established for 2-DE of embryogenic culture proteins of Gossypium hirsutum ,cv. Coker 201, by optimizing a phenol extraction method combined with methanol/ammonium acetate precipitation and optimized lysis buffer. The protein extraction for 2-DE was remarkably improved by the combination of chemically and physically modified processes including polyvinylpolypyrrolidone (PVPP) addition, acetone cleaning and TCA/acetone washes followed by aqueous methanol/ammonium acetate and acetone lavation before phenol extraction. The protocol gave a higher protein yield and higher resolution and spot intensity in 2-DE analysis. Furthermore, three lysis buffers for 2-DE were compared on cotton embryogenic calli proteins isolation. Incorporation of chaotrope mixture (7 mol¡¤L-1 urea, 2 mol¡¤L-1 thiourea) with 4% CHAPS was found to be the most effective step.
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