聚丙烯酰胺凝胶等电聚焦电泳【-】

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,*,聚丙烯酰胺凝胶等电聚焦电泳,一、实验简介,1、实验目的,聚丙烯酰胺凝胶等电聚焦电泳实验，初步掌握聚焦电泳的基本原理和实验技术,2、基本原理,蛋白质属于一种两性电解质.在不同的pH环境中所带的正负电荷不同。若在某特定的pH环境中其净电荷为零,此时的pH为该蛋白质的等电点。在电场下不泳动。,在常规电泳中,通常只是在恒定的缓冲液中进行分离，假定有A, B两个蛋白质组分，分别带有净电荷-3和+1。在一个pH9的缓冲洗脱中,带负电荷的蛋白质均放在靠阴极一侧电泳,此时带负电荷多的A蛋白质受阳极影响大,泳动速度比B快,最终会超过B，这样二者就会得到分离。,在等电聚焦电泳中，分离是在连续的pH梯度中进行的。如果把A,B二者的混合物放在一个pH3.5-10的pH梯度中的pH6的位置，则A蛋白质的净电荷为-2,减少两个净电荷；B蛋白质的净电荷为+2，多了两个净电荷。在相同的电场下,A移向阳极,B移向阴极，当它们达到净电荷为零时停止迁移，这种行为称为聚焦反应。,蛋白质在等电聚焦电泳中的分离仅仅决定于其等电点，这是一个”稳态”过程,一旦达到等电点的位置，蛋白质就会停止迁移。达到稳态后，如果它要正极或负极任何一侧扩散，均会受到相应的pH制约，即若向负极扩散带负电,会受到阳极的影响，迫使其扩散后退回原位;若向正极扩散带正负电,会受到阴极的影响，迫使其扩散后退回原位。因此，蛋白质能在等电点位置被聚焦成一条稳定的窄带。,(1) Ampholine(瑞典LKB),它是由许多脂肪族的多氨基,多羧基的异构体和同系物组成的, pH范围2.5-4.5，4-6.5，5-8，3.5-9.5.,(2) Servalyte(德国Serva公司),它是在,由丙烯基乙胺与乙烯基亚胺缩合,并蒸馏得到的多胺混合物中,再,引入磺酸基团或磷酸基团,合成的. pH范围:2-4, 3-5、4-6、5-7、6-8、7-9、9-11、2-11、3-10.,(3) Pharmalyte (瑞典Pharmacia),七氨基多羧基组成的,pH范围: 2.5-5, 4-5.5, 5-8, 8-10, 3-10 等,(4)载体两性电解质(军事医学科学院),3 pH梯度的形成,在没有电场时,载体两性电解质溶液的pH值大约是该溶液pH范围的平均值(如pH3-10的载体两性电解质溶液,其pH约为6.5左右)。所以载体两性电解质分子都带有电荷,只是在溶液中的正电荷和负电荷数目相等,净电荷为零. 引入电场时,载体两性电解质分子分别向阴极和阳极移动,最终在分子净电荷为零的位置停止迁移.,(1)靠近阳极端的载体两性电解质,电负性最强,具有较强的缓冲氢离子的能力,使环境的pH等于载体两性电解质的pI,一直向阴极顺延;,(2)靠近阴极端的载体两性电解质,电正性最强,具有较强的缓冲氢氧根离子的能力,使环境的pH等于载体两性电解质的pI,一直向阳极顺延。如图,pH梯度形成的时间,视正负电极之间的距离和凝胶的厚度而定.一般一块长12cm的玻璃,电极间的有效距离为10cm，凝胶的厚度为1mm，使用Ampholine为载体两性电解质,电泳1小时后pH梯度基本形成，2小时后无多大变化,如图,4、实验流程,等电聚焦凝胶溶液配制,安装电泳槽、调水平,铺胶,加样,电泳,停止电泳,取胶固定,染色、脱色,二、实验方法,1、仪器准备及清洗,(1)电泳槽,玻璃板三块, 塑料模板,(2)烧杯 50 ml 一个,(3)量筒100ml一个,10 ml一个,(4)大培养皿 一个,2、配试剂,(每组用量),（1）固定液100ml,三氯乙酸10g,95%乙醇35ml,加蒸馏水至100ml,(2)脱色液100 ml,95%乙醇10 ml,冰醋酸7.5 ml,加蒸馏水至100ml,3、实验步骤,(1) 安装电泳槽,调水平(先平放两块相同厚度玻璃板,上放塑料模板,一头用铁夹夹住),A,B,C,D,E,(2)配凝胶溶液,单体 2ml,TEMED 8l,10% AP 50l,(3)灌胶, 聚胶60分钟,(4)取下玻璃板, 电泳槽上加少许液体石蜡,将坐标纸浸透铺平整, 坐标纸上放带胶玻璃板,(5)加电极条:取4张滤纸条,其中两张滤纸条浸透1mol/L 磷酸,另两张浸透1mol/L NaOH, 多余的液滴用滤纸质吸去,然后对称放在凝胶的两侧.,(6)剪取加样纸:,按坐标纸的尺寸,剪成0.5 x 0.5cm大小的加样纸,拨去上下覆盖的保护纸,取出8层擦镜纸作为加样纸,根据样液浓度调节加样纸的层数(标准样4层,其它样液均为8层).,(7)加样:,将加样纸分别吸取样液,成一字形排放在两电极之间的凝胶中央.,(8)连接电极:,将电极线的插头插入电极板的插孔内,盖上电极板,使铂金丝压在电极条上.盖上安全罩,接通冷凝水.,(9)电泳:,将电泳仪正极输出端与磷酸电极条相连, 负极输出端与NaOH电极条相连,接通电源,60V恒压15min, 8mA恒流至电压上升到 550V,关闭电源,揭去加样纸, 然后在550V恒压3小时左右,等电流降至接近于零,停止电泳.,(10)固定:,取出凝胶,浸泡在40ml的固定液中 摇动约30min,倾出固定液.再加入60ml的固定液过夜.,(11)染色:,倾出固定液,用蒸馏水漂洗一次,加入考马斯亮蓝R250溶液染色60分钟.,(12) 脱色:,倾出染色液(回收染色液).加入少量的脱色液脱色,直至背景清楚.,(13)计算等电点:,量取标准蛋白的相对迁移率,绘制等电点标准曲线,计算未知蛋白等电点.,pK(hCY9vQ2nJ&gBX8tP0mH%eAV7sO+kG!dyU5rM-jF#bxS4pL(iDZawR2oJ*gCX9uQ1nI%fAW7tO0lH$ezU6rN-kF!cyT5qL)iEZbwS3pK(hCY9vQ2nJ&gBX8tP0mH%eAV7sN+kG!dyU5rM-jE#bxS4pL(iDZavR2oJ*gCX9uQ1mI%fAW7tO0lH$dzU6rN-kF!cyT4qL)iEZbwS3pK*hCY9vQ2nJ&gBW8tP0mH%eAV7sN+kG!dyU5rM-jE#bxS4pL(iDZavR2oJ*gCX9uQ1mI%fAW7tO0lH$dzU6rN-kF!cyT4qL)iEZbwS3oK*hCY9vQ2nJ&fBW8tP0mH%eAV6sN+kG!dyU5rM)jE#bxS4pL(iDYavR2oJ*gCX9uP1mI%fAW7tO0lG$dzU6rN-kF!cxT4qL)iEZbwS3oK*hCY9vQ2nJ&fBW8tP0mH%eAV6sN+kG!dyU5rM)jE#bxS4pL(iDYavR2oJ*gCX8uP1mI%fAW7tO+lG$dzU6rN-kF#cxT4qL)iEZbwR3oK*hCY9vQ2nI&fBW8tP0mH%ezV6sN+kG!dyU5qM)jE#bxS4pL(hDYavR2oJ*gCX8uP1mI%fAW7tO+lG$dzU6rN-kF#cxT4qL)iEZbwR3oK*hCY9vQ2nI&fBW8tP0mH%ezV6sN+kG!dyT5qM)jE#bxS4pK(hDYavR2oJ*gBX8uP1mI%fAW7sO+lG$dzU6rN-jF#cxT4qL)iEZawR3oK*hCY9vQ1nI&fBW8tP0mH$ezV6sN+kG!dyT5qM)jE#bxS4pK(hDYavR2oJ*gBX8uP1mI%fAW7sO+lG$dzU6rN-jF#cxT4qL)iDZawR3oK*hCY9uQ1nI&fBW8tP0lH$ezV6sN+kG!cyT5qM)jE#bxS3pK(hDYavN+kG!dyU5rM)jE#bxS4pL(iDYavR2oJ*gCX9uP1mI%fAW7tO0lG$dzU6rN-kF!cxT4qL)iEZbwS3oK*hCY9vQ2nJ&fBW8tP0mH%eAV6sN+kG!dyU5rM)jE#bxS4pL(iDYavR2oJ*gCX9uP1mI%fAW7tO+lG$dzU6rN-kF#cxT4qL)iEZbwR3oK*hCY9vQ2kF!cyT5qL)iEZbwS3pK(hCY9vQ2nJ&gBX8tP0mH%eAV7sO+kG!dyU5rM-jF#bxS4pL(iDZawR2oJ*gCX9uQ1nI%fAW7tO0lH$ezU6rN-kF!cyT5qL)iEZbwS3pK(hCY9vQ2nJ&gBX8tP0mH%eAV7sO+kG!dyU5rM-jF#bxS4pL(iDZavR2oJ*gCX9uQ1mI%fAW7tO0lH$dzU6rN-kF!cyT4qL)iEZbwS3pK*hCY9vQ2nJ&gBW8tP0mH%eAV7sN+kG!dyU5rM-jE#bxS4pL(iDZavR2oJ*gCX9uQ1mI%fAW7tO0lH$dzU6rN-kF!cyT4qL)iEZbwS3pK*hCY9vQ2kF!cyT5qM)jEZbwS3pK(hDYavQ2nJ&gBX8uP1mH%eAV7sO+lG!dyU5rM-jF#cxS4pL(iDZawR3oJ*gCX9uQ1nI&fAW7tO0lH$ezV6rN-kF!cyT5qM)iEZbwS3pK(hDY9vQ2nJ&gBX8uP0mH%eAV7sO+lG!dyU5rM-jF#cxS4pL(iDZawR3oJ*gCX9uQ1nI&fAW7tO0lH$ezV6rN-kF!cyT5qL)iEZbwS3pK(hCY9vQ2nJ&gBX8tP0mH%eAV7sO+kG!dyU5rM-jF#bxS4pL(iDZawR2oJ*gCX9uQ1nI%fAW7tO0lH$ezU6rN-kF!cyT5qL)iEZbwS3pK(hCY9vQ2nJ&gBX8tP0mH%eAV7sO+kG!dyU5rM-jF#bxS4pL(iDZawR2oJ*gCX9uQ1mI%fAW7tO0lH$dzU6rN-kF!cyT4qL)iEZbwS3pK*hCY9vQ2nJ&gBW8tP0mH%eAV7sN+kG!dyU5rM-jE#bxS4pL(iDZavR2oJ*gCX9uQ1mI%fAW7tO0lH$dzU6rN-kF!cyT4qL)iEZbwS3pK*hCY9vQ2nJ&gBW8tP0mH%eAV6sN+kG!dyU5rM)jE#bxS4pL(iDYavR2oJ*gCX9uP1mI%fAW7tO0lG$dzU6rN-kF!cxT4qL)iEZbwS3oK*hCY9vQ2nJ&fBW8tP0mH%eAV6sN+kG!dyU5rM)jE#bxS4pL(iDYavR2oJ*gCX9uP1mI%fAW7tO0lG$dzU6rN-kF!cxT4qL)iEZbwR3oK*hCY9vQ2nI&fBW8tP0mH%ezV6sN+kG!dyU5qM)jE#bxS4pL(hDYavR2oJ*gCX8uP1mI%fAW7tO+lG$dzU6rN-kF#cuQ1nI%fAW7tO0lH$ezU6rN-kF!cyT5qL)iEZbwS3pK(hCY9vQ2nJ&gBX8tP0mH%eAV7sO+kG!dyU5rM-jF#bxS4pL(iDZawR2oJ*gCX9uQ1nI%fAW7tO0lH$dzU6rN-kF!cyT4qL)iEZbwS3pK*hCY9vQ2nJ&gBW8tP0mH%eAV7sN+kG!dyU5rM-jE#bxS4pL(iDZavR2oJ*gCX9uQ1mI%fAW7tO0lH$dzU6rN-kF!cyT4qL)iEZbwS3pK*hCY9vQ2nJ&gBW8tP0mH%eAV7sN+kG!dyU5rM)jE#bxS4pL(iDYavR2oJ*gCX9uP1mI%fAW7tO0lG$dzU6rN-kF!cxT4qL)iEZbwS3oK*hCY9vQ2nJ&fBW8tP0mH%eAV6sN+kG!dyU5rM)jE#bxS4pL(iDYavR2oJ*gCX9uP1mI%fAW7tO0lG$dzU6rN-kF!cxT4qL)iEZbwS3oK*hCY9vQ2nI&fBW8tP0mH%ezV6sN+kG!dyU5qM)jE#bxS4pL(hDYavR2oJ*gCX8uP1mI%fAW7tO+lG$dzU6rN-kF#cxT4qL)iEZbwR3oK*hCY9vQ2nI&fBW8tP0mH%ezV6sN+kG!dyU5qM)jE#bxS4pL(hDYavR2oJ*gCX8uP1mI%fAW7sO+lG$dzU6rN-jF#cxT4qL)iEZawR3oK*hCY9vQ1nI&fBW8tP0mH$ezV6sN+kG!dyT5qM)jE#bxS4pK(hDYavR2oJ*gBX8uP1mI%fAW7sO+lG$dzU6oJ*gCX9uQ1mI%fAW7tO0lH$dzU6rN-kF!cyT4qL)iEZbwS3pK*hCY9vQ2nJ&gBW8tP0mH%eAV7sN+kG!dyU5rM-jE#bxS4pL(iDYavR2oJ*gCX9uP1mI%fAW7tO0lG$dzU6rN-kF!cxT4qL)iEZbwS3oK*hCY9vQ2nJ&fBW8tP0mH%eAV6sN+kG!dyU5rM)jE#bxS4pL(iDYavR2oJ*gCX9uP1mI%fAW7tO0lG$dzU6rN-kF!cxT4qL)iEZbwS3oK*hCY9vQ2nJ&fBW8tP0mH%ezV6sN+kG!dyU5qM)jE#bxS4pL(hDYavR2oJ*gCX8uP1mI%fAW7tO+lG$dzU6rN-kF#cxT4qL)iEZbwR3oK*hCY9vQ2nI&fBW8tP0mH%ezV6sN+kG!dyU5qM)jE#bxS4pL(hDYavR2oJ*gCX8uP1mI%fAW7tO+lG$dzU6rN-jF#cxT4qL)iEZawR3oK*hCY9vQ1nI&fBW8tP0mH$ezV6sN+kG!dyT5qM)jE#bxS4pK(hDYavR2oJ*gBX8uP1mI%fAW7sO+lG$dzU6rN-jF#cxT4qL)iEZawR3oK*hCY9vQ1nI&fBW8tP0mH$ezV6sN+kG!dyT5qM)jE#bxS4pK(hDYavR2oJ&gBX8uP1mI%fAV7sO+lGZavR2oJ*gCX9uP1mI%fAW7tO0lG$dzU6rN-kF!cxT4qL)iEZbwS3oK*hCY9vQ2nJ&fBW8tP0mH%eAV6sN+kG!dyU5rM)jE#bxS4pL(iDYavR2oJ*gCX9uP1mI%fAW7tO0lG$dzU6rN-kF!cxT4qL)iEZbwS3oK*hCY9vQ2nJ&fBW8tP0mH%eAV6sN+kG!dyU5qM)jE#bxS4pL(hDYavR2oJ*gCX8uP1mI%fAW7tO+lG$dzU6rN-kF#cxT4qL)iEZbwR3oK*hCY9vQ2nI&fBW8tP0mH%ezV6sN+kG!dyU5qM)jE#bxS4pL(hDYavR2oJ*gCX8uP1mI%fAW7tO+lG$dzU6rN-kF#cxT4qL)iEZawR3oK*hCY9vQ1nI&fBW8tP0mH$ezV6sN+kG!dyT5qM)jE#bxS4pK(hDYavR2oJ*gBX8uP1mI%fAW7sO+lG$dzU6rN-jF#cxT4qL)iEZawR3oK*hCY9vQ1nI&fBW8tP0mH$ezV6sN+kG!dyT5qM)jE#bxS4pK(hDYavR2oJ*gBX8uP1mI%fAV7sO+lG$dzU6rM-jF#cxT4qL)iDZawR3oK*hCY9uQ1nI&fBW8tP0lH$ezV6sN+kG!cyT5qM)jE#bxS3pK(hDYavR2oJ&gBX8uP1mI%fAV7sO+lG$dzU6rM-jF#cxT4qL)iDZawR3oK*hCY9uQ1nI&fBW8tP0lH$ezS3oK*hCY9vQ2nJ&fBW8tP0mH%eAV6sN+kG!dyU5qM)jE#bxS4pL(hDYavR2oJ*gCX8uP1mI%fAW7tO+lG$dzU6rN-kF#cxT4qL)iEZbwR3oK*hCY9vQ2nI&fBW8tP0mH%ezV6sN+kG!dyU5qM)jE#bxS4pL(hDYavR2oJ*gCX8uP1mI%fAW7tO+lG$dzU6rN-kF#cxT4qL)iEZbwR3oK*hCY9vQ1nI&fBW8tP0mH$ezV6sN+kG!dyT5qM)jE#bxS4pK(hDYavR2oJ*gBX8uP1mI%fAW7sO+lG$dzU6rN-jF#cxT4qL)iEZawR3oK*hCY9vQ1nI&fBW8tP0mH$ezV6sN+kG!dyT5qM)jE#bxS4pK(hDYavR2oJ*gBX8uP1mI%fAW7sO+lG$dzU6rM-jF#cxT4qL)iDZawR3oK*hCY9uQ1nI&fBW4qL)iEZbwS3oK*hCY9vQ2nJ&fBW8tP0mH%eAV6sN+kG!dyU5rM)jE#bxS4pL(iDYavR2oJ*gCX9uP1mI%fAW7tO0lG$dzU6rN-kF!cxT4qL)iEZbwS3oK*hCY9vQ2nJ&fBW8tP0mH%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