为探索苹果叶片蛋白质SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析的样品制备方法,比较了三氯乙酸(TCA)/丙酮沉淀法、改良的Tris-HC1法、酚-甲醇/醋酸铵沉淀法、二硫苏糖醇(DTT)/丙酮法及Tris-HC1法等5种蛋白质的提取方法.制备的样品经SDS-PAGE分离采用银染显色.结果表明,改良的Tris-HC1法在加大PVPP用量和蛋白质提取缓冲液的基础上,将丙酮沉淀蛋白质的时间由30 min增加到1 h,使蛋白质的得率最高为3.23μg/μL,上样量5μL得到的蛋白质条带数量最多,条带最清晰为1条.利用这一改良方法对苹果实生树不同节位蛋白质的动态变化进行了研究,共发现6条蛋白质差异带,分子量分别为71.9,60.5,52.6,1.1,35.3,18.5 kDa,条带清楚,背景清晰.因此,改良的Tris-HC1法适用于苹果叶片的SDS-PAGE分析.
曾广娟
,
李春敏
,
张新忠
,
陈东玫
,
赵永波
,
董文轩
. 适于SDS-PAGE分析的苹果叶片蛋白质提取方法[J]. 华北农学报, 2009
, 24(2)
: 75
-78
.
DOI: 10.7668/hbnxb.2009.02.016
In order to develop an efficient protein extraction method suitable for SDS - PAGE analysis,five protocols for protein extraction in apple leaf,trichloroacetic acid/acetone precipitation (TCA),modified Tris - HCl extraction,phenol extraction methanol/ ammonium acetate precipitation,DTT/ acetone and Tris - HCl extraction were compared.The extracts were separated by SDS - PAGE method followed by silver staining. The results showed that the modified Tris-HCl extraction presented the highest resolution of 3. 243μg/μL and the maximum bands of 41 strips with the 5μL sample volumes basing on the increased amount of PVPP and the extracting solution as well as prolonged 30 min of protein precipitation with cold acetone.Dynamic changes of proteins in leaves from different nodes in apple seedlings (Jonathan ×Golden Delicious)were analyzed with the improved Tris - HCl extraction method,six protein bands were detected with the molecular weight 71.9,60.5,52.6,41.1,35.3,18.5 kDa respectively and the bands were clear with a light background.So the improved Tris - HCl extraction was the most appropriate method for apple leaf SDS - PAGE analysis。
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