采用水平双相凝胶电泳法对福赛斯拟杆菌蛋白分离的初步研究
Separation ofBacteroides ForsythusATCC43037 Proteins by Horizontal Two-Dimensional Gel Electrophoresis
作者：黄定明1,周学东1,久保庭雅惠2,天野敦雄2
Author：HUANG Ding-ming1,ZHOUXue-dong1,KUBONIWAMasae2,AMANO Atsuo2
收稿日期：2004-06-25 年卷（期）页码：2004,22(03):177-
期刊名称：华西口腔医学杂志
Journal Name：West China Journal of Stomatology
关键字：水平双相凝胶电泳,福赛斯拟杆菌,蛋白分离,
Key words：horizontal two-dimensional electrophoresis,Bacteriodesforsythus,protein separation,
基金项目：
四川省科研基金资助项目(03JY029-089-1)
中文摘要
目的 为分离福赛斯拟杆菌蛋白,建立一种快速、方便、高效、可靠的方法。方法 福赛斯拟杆菌厌氧培养的菌细胞经超声破碎后,菌蛋白加入固相pH梯度(IPG)膨润液中或经ReadyPrep试剂处理后,在不使用加样杯、加样杯分别位于IPG的阳极端、中间、阴极端,采用pH3~10的IPG聚丙烯酰胺凝胶条作等电聚焦电泳,梯度为8% ~18%聚丙烯酰胺板状梯度凝胶进行第二相电泳,经考马斯亮兰染色或蛋白银染观察其蛋白斑点。结果 ①等电聚焦电泳时,未使用加样杯福赛斯拟杆菌蛋白加入膨润液中双相电泳后两种染色都未见蛋白斑点,用加样杯加入福赛斯拟杆菌蛋白经水平双相电泳后考马斯亮兰染色无斑点出现而银染出现蛋白斑点,且加样杯位于IPG中间位置时蛋白等电聚焦的效果最佳。②福赛斯拟杆菌蛋白电泳图谱显示水平双相凝胶电泳能将不同蛋白分离,且绝大多数蛋白位于IPG的酸性端,即蛋白的等电点(PI)在3~7之间。结论 采用水平双相凝胶电泳方法可以将福赛斯拟杆菌不同种类的蛋白分离。
英文摘要
Objective To set up a rapid, efficient, reliable and accurate method for separation ofBacteriodesforsythuspro- teins.Methods BacteroidesforsythusATCC43037 cells were harvested by centrifugation, washed inTris buffer to remove excess medium, and lysed by sonication. The sonicated lysis proteins were extracted step by step with ReadyPrepTMSequential Extraction Kit (Bio-Rad). The supernatant ofB.forsythusproteins were used for two-dimensional gel electrophoresis. The first dimension IEF(isoelectric focusing) was run with Immobiline DryStrip (pH 3~10) and the second dimension SDS-PAGE was run with Ex- celgel SDS,gradient 8-18 precast gel and buffer strips. The separated proteinswere stainedwith Coomassie Brilliant Blue and silver staining kit (Plusone Silver Staining Kit, Protein, Pharmacia Biotech).Results 1. The protein spots were clear when sample cups were used in the middle of IPG strip during IEF. 2.B.forsythusproteinswere separated clearly by horizontal two-dimension- al electrophoresis and most ofB.forsythuswhole-cell proteins were acidic proteins (PI3-7).Conclusion Horizontal two-dimen- sion electrophoresis is a useful method for separatingB.forsythusproteins.
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