请问请问如果蛋白凝胶电泳法电泳主副槽相通,会影响电泳效果吗?

2025-03-30 11:27:22
推荐回答(1个)
回答1:

如果蛋白凝胶电泳法电泳主副槽相通,会影响电泳效果
(1)由于与凝胶聚合有关的硅橡胶称、玻璃板表面不光滑洁净,在电泳时会造成凝胶板与玻璃板或硅橡胶条剥离,产生气泡或滑胶;剥胶时凝胶板易断裂,为防止引现象,所用器材均应严格地清洗.硅橡胶条的凹槽、样品槽模板及电泳槽用泡沫海绵蘸取“洗洁净”仔细清洗.玻璃板浸泡在重铬酸钾洗液3~4h或0.2mol/L KOH的酒精溶液中20min以上,用清水洗净,再用泡沫海绵蘸取“洗洁净”反复刷洗,最后用蒸馏水冲洗,直接阴干或用乙醇冲洗后阴干.
(2)安装电泳槽和镶有长、短玻璃板的硅橡胶框时,位置要端正,均匀用力旋紧固定螺丝,以免缓冲液渗漏.样品槽板梳齿应平整光滑.
(3)在不连续电泳体系中,预电泳只能在分离胶乡合后进行.洗净胶面后才能制备浓缩胶.浓缩胶制备后,不能进行预电泳,以充分利用浓缩胶的浓缩效应.
(4)电泳时,电泳仪与电泳槽间正、负极不能接错,以免样品反方向泳动,电泳时应选用合适的电流、电压,过高或过低无可影响电泳效果.
(5)SDS纯度:在SDS-PAGE中,需高纯度的SDS,市售化学纯SDS需重结晶一次或两次方可使用.重结晶方法如下:称20g SDS放在圆底烧瓶中,加300ml无水乙醇及约半牛角匙活性碳,在烧瓶上接一冷凝管,在水浴中加热至乙醇微沸,回流约10min,用热布氏漏斗趁热过滤.滤液应透明,冷却至室温后,移至-20℃冰箱中过夜.次日用预冷的布氏漏斗抽滤,再用少量-20℃预冷的无水乙醇洗涤白色沉淀3次,尽量抽干,将白色结晶置真空干燥器中干燥或置40℃以下的烘箱中烘干.
(6)用SDS处理蛋白质样品时,每次都会在沸水溶中保温3~5min,以免有亚稳聚合物存在.
(7)标准蛋白质的相对迁移率最好在0.2~0.8之间均匀分布.值得指出的是,每次测定未知物分子量时,都应同时用标准蛋白制备标准曲线,而不是利用过去的标准曲线.用此法测定的分子量只是它们的亚基或单条肽链的分子量,而不是完整的分子量.为测得精确的分子量范围,最好用其他测定蛋白分子量的方法加以校正.此法对球蛋白及纤维状蛋白的分子量测定较好,对糖蛋白,胶原蛋白等分子量测定差异较大.
(8)对样品的要求:应采纳低离子强度的样品.如样品中离子强度高,则应透析或经离子交换除盐.加样时,应保持凹形加样槽胶面平直.加样量以10~15μl为宜,如样品系较稀的液体状,为保证区带清晰,加样量可增加,同时应将样品溶解液浓度提高二倍或更高.
(9)由于凝胶中含SDS,直接制备干板会产生龟裂现象.如需制干板,则用25%异丙醇内含7%乙酸浸泡,并经常换液,直到SDS脱尽(约需2~3天),才可制备干板.为方便起见,常采用照像法,保存照片.

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