CN106644657A - 4’,5’‑二溴荧光素及其衍生物在蛋白检测中的应用 - Google Patents
4’,5’‑二溴荧光素及其衍生物在蛋白检测中的应用 Download PDFInfo
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Abstract
本发明涉及蛋白质负染检测技术，具体地说是4′，5′‑二溴荧光素及其衍生物在蛋白质负染检测中的应用。本发明还提供了应用4′，5′‑二溴荧光素进行蛋白负染检测的方法，包括步骤：蛋白质凝胶电泳结束后直接染色；接着显影。本发明具有灵敏度高、操作简单、重现性好、质谱兼容性好和成本低廉等优点，可较好适用于高通量蛋白质组学的研究。
Description
技术领域
本发明涉及蛋白质负染检测技术，具体地说，涉及蛋白质检测的一种新的负染染料。
背景技术
在蛋白质组学研究中，凝胶蛋白质染色技术是衔接二维电泳和下游质谱分析的关键技术。当前先进的质谱仪已能够分析pg级别的痕量蛋白。常规的凝胶蛋白质染色技术有考马斯亮蓝染色、银染、荧光染色等。考马斯亮蓝染色法灵敏度低，银染法操作复杂、质谱兼容性差，荧光染色法价格昂贵。这些因素严重地制约了高通量蛋白质组学的发展。虽然，近年来开发了不少新的染色技术，但仍存在着如下一个或几个缺点：灵敏度低、重现性差、毒性大、质谱兼容性差、价格昂贵、操作繁琐等。寻找新的染色方法以适应当前先进的质谱分析技术已成为后基因组时代推动蛋白质组学发展的当务之急。4′，5′-二溴荧光素为凝胶蛋白染色提供了一种快速、灵敏的方法，其灵敏度达0.025ng/band，可以和银染相媲美。而且4′，5′-二溴荧光素负染方法是对凝胶背景进行着色，对蛋白质的结构没有修饰或破坏作用，有良好的质谱兼容性，弥补了银染法质谱兼容性差的缺陷。4′，5′-二溴荧光素负染将很好地衔接二维电泳和下游质谱分析，推动蛋白质组学的快速发展。
发明内容
本发明的目的在于提供4′，5′-二溴荧光素及其衍生物在蛋白质检测中的应用。
本发明所述的相关化合物是指以4′，5′-二溴荧光素阴离子为母核的钠盐、钾盐和铵盐等。
4′，5′-二溴荧光素母核为：
为实现本发明的目的，本发明提供了一种电泳凝胶上蛋白质负染的方法，包括如下步骤：
1)将经SDS-PAGE电泳后的蛋白质样品置于染色液中染色5～15min，其中染色液为含重量体积比0.05～0.5％的4′，5′-二溴荧光素或其衍生物、按摩尔浓度比20～100μM的氯化锌、按溶液体积比5～20％的甘油及按溶液体积比30％～70％的甲醇水溶液；优选的染色时间为8min，优选的4′，5′-二溴荧光素及其衍生物的浓度为0.3％，优选的氯化锌浓度为50μM，优选的甘油浓度为10％，优选的碳酸优选的甲醇浓度为40％。
2)弃去染色液，加入显影液显影1～5min，其中显影液为按摩尔浓度比100mM的醋酸钠-醋酸缓冲液，pH＝4.6。优选的显影时间为2min。
3)检测，凝胶染色后，可放在黑色背景下直接肉眼观察；或在爱普生V700扫描仪下，反向扫描模式扫描，其灵敏度更高。
前期研究表明该技术有如下优点：
1)灵敏度高：4′，5′-二溴荧光素负染灵敏度高于考马斯亮蓝染色法数百倍，和银染法灵敏度相当；
2)操作简单迅速：操作步骤少，可在10min内完成；
3)重现性好：受温度、摇床摆动频率等外部条件影响小；
4)可逆性好：容易脱色；
5)质谱兼容性好：由于4′，5′-二溴荧光素负染是对凝胶背景进行染色，不和凝胶上的蛋白质结合，蛋白质结构完全不受影响，所以可与LC-MS等质谱仪高度兼容；
6)使用安全：采用毒性低的染料，提高实验操作的安全性，对环境污染小；
7)成本低。
附图说明：
图1. 4′，5′-二溴荧光素的化学结构。
图2. 4′，5′-二溴荧光素负染法染色和显影条件的优化。(A)染色液中4′，5′-二溴荧光素浓度的筛选；(B)染色液中甲醇浓度的筛选；(C)染色液中甘油浓度的筛选；(D)染色液中氯化锌浓度的筛选；(E)染色时间的筛选；(F)显影液中pH值的筛选。
图3.在一向SDS-PAGE上，(A，E，I)4′，5′-二溴荧光素负染法；(B，F，J)曙红负染法；(C，G，K)SYPRO Ruby荧光染色法；(D，H，L)锌咪唑负染法。(A-D)为Sigma公司的蛋白标准品，(E-H)为小鼠脑总蛋白，(I-L)为大肠杆菌Escherichia coli BL21总蛋白。蛋白标准品上样量如下(A-D)：带1，12.8ng；带2，6.4ng；带3，3.2ng；带4，1.6ng；带5，0.8ng；带6，0.4ng；带7，0.2ng；带8，0.1ng；带9，0.05ng；带10，0.025ng。小鼠脑总蛋白和大肠杆菌Escherichiacoli BL21总蛋白上样量(E-L)从左至右倍倍稀释。
图4.在二向SDS-PAGE胶上，(A)4′，5′-二溴荧光素负染法和(B)锌咪唑负染法灵敏度的比较。分离样品小鼠脑总蛋白；IPG胶条长13厘米，pH4-7；分离胶浓度为11.4％；样品上样量为100微克/胶条。(C)4′，5′-二溴荧光素负染法和锌咪唑负染法检测到的蛋白斑点数统计。
具体实施方式
下面结合具体地实施例来进一步阐述本发明。应当理解，这些实施例仅用于说明本发明，而不能限制本发明的保护范围。
实施例1 4′，5′-二溴荧光素负染染色
图1是4′，5′-二溴荧光素的化学结构式。
图2～4的4′，5′-二溴荧光素蛋白质负染实验采用下述步骤进行：
1)将电泳后的凝胶置于0.3％4′，5′-二溴荧光素/40％甲醇/10％甘油/50μM氯化锌染色液中染色8min。
2)弃去染色液，在100mM的醋酸钠-醋酸缓冲液(pH＝4.6)中显影2min。
3)凝胶染色后，在爱普生V700扫描仪下，反向扫描模式扫描。
按照上述方法分别用4′，5′-二溴荧光素的钠盐、钾盐、铵盐进行蛋白质染色，结果表明用这些衍生物均能够得到类似于4′，5′-二溴荧光素的检测结果。
图2是4′，5′-二溴荧光素负染法染色法条件优化的过程。分别对染色液中4′，5′-二溴荧光素浓度、甲醇浓度、甘油浓度、氯化锌浓度、染色时间及显影液中pH进行了筛选。结果显示最佳的染色液条件为0.3％4′，5′-二溴荧光素/40％甲醇/10％甘油/50μM氯化锌，最佳染色时间为8min，显影液最佳pH值为4.6。
图3是说明在一向SDS-PAGE上，4′，5′-二溴荧光素负染法与其它染色法效果的对比，采用的是蛋白标准品，小鼠脑总蛋白和大肠杆菌Escherichia coli BL21总蛋白。
按照实施例1的方法，采用不同的方法进行染色，(A，E，I)4′，5′-二溴荧光素负染法；(B，F，J)曙红负染法；(C，G，K)SYPRO Ruby荧光染色法；(D，H，L)锌咪唑负染法灵敏度的比较。曙红负染法、SYPRO Ruby荧光染色法和锌咪唑负染法均按照文献记载。蛋白标准品上样量如下(A-D)：带1，12.8ng；带2，6.4ng；带3，3.2ng；带4，1.6ng；带5，0.8ng；带6，0.4ng；带7，0.2ng；带8，0.1ng；带9，0.05ng；带10，0.025ng。小鼠脑总蛋白和大肠杆菌Escherichiacoli BL21总蛋白上样量(E-L)从左至右倍倍稀释。结果如图3所示，4′，5′-二溴荧光素负染法灵敏度最高。
小鼠脑总蛋白提取方法如下：用4℃的PBS溶液清洗小鼠脑组织，一共清洗3次。之后用液氮进行组织研磨，将研磨所得的粉末装入事先称重的EP管中。向EP管中加入裂解液(9.5M urea，0.1DTT，2％CHAPS，0.8％pharmalyte pH3-10)至终浓度为1mg/mL。将样品超声破碎3次，每次1分钟，完毕之后15000rpm 4℃高速离心30min，取上清液。大肠杆菌总蛋白的提取方法如下：取Escherichia coli BL21菌液，12000rpm离心收集菌群，加入0.02mol/L的磷酸盐缓冲液(pH＝7.0)重悬菌体。利用超声破碎仪破碎细胞。18000rpm离心30min，收集上清，即得Escherichia coli BL21总蛋白质提取液(粗蛋白制品)。
图4是说明在二向SDS-PAGE上，4′，5′-二溴荧光素负染法与锌咪唑负染法效果的对比，采用是小鼠脑总蛋白。
用4′，5′-二溴荧光素负染法和锌咪唑负染法对经二向凝胶电泳分离的小鼠脑总蛋白进行染色，结果如图4所示。结果表明，相比较锌咪唑负染法而言，4′，5′-二溴荧光素负染法可检测到更多的蛋白质斑点，具有更高的灵敏度。
图2～4中的其它染色方法及相关文献
曙红负染法操作方法参考文献：Cong WT，Hwang SY，Jin LT，He HZ，ChoiJK.High-throughput negative detection of SDS-PAGE separated proteins and itsapplication for proteomics.Electrophoresis 2010，3，411-420.
SYPRO Ruby荧光染色法参考文献：Berggren K，Chernokalskaya E，SteinbergTH，Kemper C，Lopez MF，Diwu Z，Haugland RP，PattonWF.Background-free，highsensitivity staining of proteins in one-and two-dimensional sodium dodecylsulfate-polyacrylamide gels using a luminescent rutheniumcomplex.Electrophoresis 2000，21，2509-2521.
锌咪唑负染法操作方法参考文献：Castellanos-Serra L，Proenza W，Huerta V，Moritz RL，Simpson RJ.Proteome analysis of polyacrylamide gel-separatedproteins visualized by reversible negative staining using imidazole-zincsalts.Electrophoresis 1999，20，732-737。
Claims (6)
1.4′，5′-二溴荧光素及其衍生物在蛋白质检测中的应用。
2.如权利要求1所述的应用，其特征在于所述的4′，5′-二溴荧光素及其衍生物为4′，5′-二溴荧光素的钠盐、钾盐或铵盐。
3.一种凝胶蛋白负染检测方法，其包括步骤：
1)将经SDS-PAGE电泳后的蛋白质样品置于染色液中染色5～15min，其中染色液为含重量体积比0.05～0.5％的4′，5′-二溴荧光素或其衍生物、按摩尔浓度比20～100μM的氯化锌、按溶液体积比5～20％的甘油及按溶液体积比30％～70％的甲醇水溶液；
2)加入显影液显影1～5min，其中显影液为按摩尔浓度比100mM的醋酸钠-醋酸缓冲液，pH＝4.6。
3)检测。
4.如权利要求3所述的方法，其特征在于，步骤1)中染色时间为8min，染色液为0.3％4′，5′-二溴荧光素/40％甲醇/10％甘油/50μM氯化锌。
5.如权利要求3所述的方法，其特征在于，步骤2)中显影时间为2min。
6.如权利要求3所述的方法，其特征在于，步骤3)检测即可用肉眼直接观察，又可以用爱普生V700扫描仪反向扫描模式扫描。
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