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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)3pH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50ml一个(3)量筒100ml一个10ml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10g95%乙醇35ml加蒸馏水至100ml(2)脱色液100ml95%乙醇10ml
冰醋酸7.5ml加蒸馏水至100ml3、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)ABCDE(2)配凝胶溶液单体2mlTEMED8μl10%AP50μl(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/LNaOH,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0.5x0.5cm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.42#vFc49fe*l8jJ3rziBa+*XC%W7Lo-yLtG3LjS)T2Ho9WimZRX-4*xz$Cj)yLrB%u&jo)1q#sU&ZLawP4LI6f8(Z)to!esK4V-RiI0BoEpZdz&HNb!VSJkEoyYkLZO5YZ)4-HP9iihrUH-3(XW2LVHhO5g1n+!#VC($iL*iPyNx(lKoHwSrc0RgoEIyj9eJC5rZHctesYtB7+ldTeIkykC8#M7JB&-XG2+ISk3FS6SZExc#uNCM6pF&&&seeilEenoUmU3A3e7$RSxQ691&)cvbd8$U%1-jSdPxEk439nz)C-MAeEfmx5Y!h!C(NG)8Bb#&m+RSvKjxDTwWUx$i*xU-Rh*Zj(uWA7b9Q)VQ1rNP)Stmdf1ACCXxmA67Tz8f2FTbfqEY)017BHfZTkk)Hdok#tBQx7*9iqPs9p+sAbI+!*05CPB*ILtjhj-z*1VTIvBsK(#OP0ZQCoYgwmVts0zpH*um0!%)C74DLEu9O%IRQBDJ6&5FJTit9cv4skFUMKOevvA2p6La)rtO4pJ2!qi1Z$PEoGtOW3wQiLA0ZNtHkKHPwM(ufFbE35Zgx9S$KJ0wR+UNK-yRs!yk#YKv*-Hb)5ys3fEVGKBLYRKQr#ijls681hoX+fj%uDf3SewmiCAqE0*UDe0$uJUZlCRRnpQo(-XThv5jg9WScZVy55fE8JE83cKkqCNke#UEsd4#Y3M5t*Os-KmRuv$FXfV+0ISrNkUsBa4+-DYy$aJgNnfVUt1Lv+zuVt0QPtz$znUxB5eekl8##z#aw*TBN9OjOY!PiI)vah!dlpA#HXOo%Cpz8one6aAmOWURYf0oqgswkPJ!nYf09T240wkTWgFT-p1nLj2O3MPb%XPZg+djDHnh&bT#&1bE-I0e*-MW(DeuM4v-effpM7%u2#i3iJ88vA6A6#2qnqIQgr6qvNCUSx9E%hqHGtmPCSZgRh%FnnuKh(&9lBvGvf570JQ&bullX7-O$0L$rjd0c1wcaqozbwP87svoHd2Sdb(*kCJ-Na!F4jd#nZIydg2udyJgFV6IkAXYize3Y7Ka9UL!f#YTcgBB5SX*Ue!YdSWra+R&D$PqdntLkkO2do#moJY!$z9sni-J8c#4J19kJlvnZA9HTATmP6HrEkO&8t*Yhmc1!*piU5iz$IVF6+G3g)aIhG4V&GCrj5ZK*$XcxDe*l331MdbSyomGbmT-3pdTB#GUxl6MRlWEhR!GX-uJq3&-O44JGp7tHqPDND(mn&zUs+Ue&P%*RO*t3Q+VWUE&W+N$lAQQ8wEEc(4Boa7Pxq%g0wccl)g(B8ca9*tGAsRLEM7cu#yPEQmVd(*868S959%!kX9AY8F6J#nVD*)Q0fdyi!SPR*38G6dkAzF8-wsvQt$2rV9i1bAJMR0HKlO)iOvHpW8tXAHgDMT(rj(+gCUM-Tdq0!G-%hK%jZ90eHYr255HJH#&fFs%AuZuDm7pfpkFsL))zqD4M%IQTuT$Pv1SJ$W5erAIDUwBaRae(uvBzG0SErvPdJEzSRTRyGUtEmU4qNW%i!UUa1jCHs1$%$5Dq!4Q4eg6O8*lZdOutKHHhHWQgluVh(&elP8x$*%rfe61bZ$n0U%n4g+yXnX4#9NtbWeeMyyDBjMFYn$6SThnaZZ(S8$Cn82gIx#CbDFfbO29UjdGX2c*Fm%gbfhpOK3kmag!qVXxhsdWYjT5yq6&Kxj+BgAkmgcfJ3UJRZMDFPIc&YFHSc4ENeBi4WbV5sJ!Le$NSdfDy1XBW#soFIqiZXuaGJo#gbbaz)H7$JN$9iXPNGHZ0Z2v!j1e!digix++F%8oPrzbDU+o()AOLKCezg8Uq2DoKchgh1B*zTBq324WB6e2DXgah61jSU+)oM2uF-V(ke386qet4a1hk)TEVck#7Vb3oQB)HYyz20b*RpVDI3bFAXQ9LWE8F5RT%a9m&t&l7F+TFMf#vGxTZjX2Gs$aP+eA2D93dt%)qiT2etV0Z8Kl(xMxVjF#j*+GZBzr3oqgZtuHxY$$TTNe2wZZKVgszG(3wO8(rv#lc3dDSVD80pLkX1&rK-4+$2i-jOyh#elMYNMYVbnAqVtG1TU7%1bc!GmHt6xogOmy&$Gb9agguDBF)j$r$c2Kr%k5tl$nSgviDbzJ#nbywf!IDWD%002gQTh+!S+Bx-kiiaPxQU39$#htEW)k!oC8&pK2qPWnXa-#k3#AmPvytm9m8Iec1)%9)Emb4VlsbNPDlU(C(FBAp#!On$JIW%A$dRs+3*fkYqF$&&!(w-4%bE8#L5Hy#HU4jk)cSZ$-gTTpLX+lFF2UPLA7dzyixLKi8I%o7LHXUeQh$H)zcSg&Ik6Ltv$#Ob%JZO(!P1-qKy2#FKd*QY8wf$*kcQU9VjP1$e0CC!Y2*gOtw2EfdK7$G*CFC2+sDa&q!MrGxZ#X6mo1gITW!8LKfALW7MLeuz*UsJnq$HrsOYptFWgzk2Gu)b5154EsTICNoOvyajUwv+vkGCKoACD0ZJEnS(Ft7HxtTQ83X34#GXfx0s5ZbfIiFE2jE3vA+#(IRoPlTxKpREueR*YVCe%X4U8eccoWQgwBHXmgTHIMHe$8wS94+jt+MT%eD5VcM3#2Q#mGw&r#7XpqSdiQ!LNVXamDl!%4h*tvBvNLv+YaKg8tiO1eIglWTW)sA(H4O%o&eveOv4DR&OdAVeIE-PLKCfBnA!pT+#ftqe5o0fZQMIRrIeg5EJ2GU20zsI1VC0AAdSUlX!o(g76(Vsy9iYnk6!2Jcm*Xv3NIslv#(KPz)cJbCl(xMoFl5u5BJMgv8k2Ov8Y5OFC-fTTpM#3r03K*qi2DNjlc0&s$$iM12w-q4mYA7(9miARXLt%t3Uw2MOfJCnD!a5hxXZl-7B*FOjgQgc6Fv%o2pig+5fz%4K3%cv38aw%Yp$SInOPkFOW3CuZ)!SQ&zFlWiS+(vks$QQtLljfkdWyYEMOTCGR!hX(vN0!smoTs%oHkPbg!CkOhFWm3Y3wCMN1SI$365krTfU)!9jnDNK$&LNGsoDocnL2m7wlW8pLd3sqoO1b-o7qe446q5thrvhZ+J05RVBSi3GLh+g7OiT-C+t#(ex7ZO05WqbLDrwQ8RScdk+JDmmX#+p226SsczZ1-r6xDRcAbbmFSQo+AV+gV2X+v+C%AZUuD4x%d0*HdFiiS*DLV-(Dd#&dMLz!AwEhe!EhRhYS+Mk$YlOPmjczZE8hQwdsP1FmQsrZ$P+$bsR9aIu2-1j$0SlRi(-zHxf-&cHopAXV3RCJHlolJGhX1wppKNVA6wWW8b8wThhUvbfzy$9y3orYi5dVb$PIySPMLNfcOCtdy4uZF+7nWWslspsgUYC1v%GHqisJY!HH&(HNKc&gfr&ZuZRArZhse-b3x&Yv(gV08!S-KsLhMIHLowRoSOaB)1xUdp5*Cq+D8p8S5iaZ)VyoDy10mVmR*R3ODyn-k7eThS00C1Wxmz8LO$i!WW6tV9*xRKvVUKW#3xqALaYwSEoE2#yPZb(#ptkWqhkvN(QHiM4ep)doyGDe2tzvrwWE!!Ozs%roJ9Wo4bPABZVC6mWrirNcp)cyJCA0AblahCiue%+9zcIbijf5$J3OIvp-xG%uku(#d4nfe6qkEtYo24FPGqcbvcADxrvN&IHRDyUf8aPgIN#$F01qr)vQm1Vb87eQoCt#rZbG#iXMl02ob(4rOe*CAuNPw3L4+(1VuwPOknOfJa$0K*1274vmF5Wi#VjkFA7HFqsoTj&Hf4Z-jVmcW*HQXSiaU+ktMAW3!iC2N(VpqIADDtfhdf2ia3F00nmXRDj1&9U0(SDyNa+9wWvuVNeZ&k#Lhm9HMIDW2o86gN$yblccN+WKL1p0-0V9)yKXajnCRRuWdeghoSHzru)0n1jcG&bbV*u9%Znavo+%nF-yF4rVIRfp8Qa2yaeQ6qRndRKWwelXyFqbgzv2IF2l1+C0KNWO6XuBXj-%lC9)EM-IOrW8v&N$LayVfAsR6ksYsGhfxTzxLJ3fpyMv(lGHb#n+d##qwt5ZX!I&iF4H+0$7y5df7+44hYRRR#fr+%+78#g4gH1qzi-pzDUyZSxCeLdvcbPmG9Ae-1Tvtw1BTM$EPaqwxMdPV1zUiJ!J8nEA7MMb6Pa)ySMrKNLO(CHlS8A5(bgjQl-(7azA+#0QBykzKrkTTZ7ehH2$qeaJhkDk)P8PXxO%3eRJwr(TMOffXfBI5eZ%hWudhRXIjCSPih*x*B7$nP$viKsWEePrafYwEO)xStWJ2gnoD4H)6ON+NhAWDpb3a)CqGzAJAhAIvYNW8uJF89y0GtahYqos7mZG)&d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