CN106632215B - 一种荧光蛋白质染色剂及其制备方法和应用 - Google Patents
一种荧光蛋白质染色剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种荧光蛋白质染色剂及其制备方法和应用，该蛋白质染色剂的结构式为
Description
技术领域
本发明属化学与生物传感技术领域，具体涉及一种具有荧光性能的蛋白质染色剂，以及该蛋白质染色剂的制备方法和应用。
背景技术
有机小分子荧光探针广泛的应用于环境科学、生命科学和材料科学等领域，并日益成为现代生命科学及疾病诊断等领域不可缺少的研究手段，因此，开发具有实用价值的功能性荧光染料分子倍受关注。以有机荧光团为基础的分子荧光探针具有灵敏度高、操作简便、重现性好、膜透性好、原位检测等众多优点。而且，荧光染料常用作荧光探针，在标记生物分子或微粒的生物工程与医疗检测领域具有重要应用。目前，许多的应用是利用荧光染料作为检测工具，其需要荧光染料与蛋白质、氨基酸、核酸及其他的生物分子共价结合形成染料标记的配体。
十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)是Shapiro于1967年建立的，经过不断的改进、完善，现在已广泛应用于蛋白质分析。由于SDS覆盖蛋白质分子，使蛋白质能够根据分子大小带有相应的负电荷，使得蛋白质的电泳迁移率不受其原有电荷影响，而只与相对分子质量有关。因此通过SDS-PAGE可分离蛋白并测定蛋白质相对分子质量和蛋白质纯度。在采用SDS-PAGE分析蛋白质特性时，考马斯亮蓝、银染等是应用最为广泛的蛋白染色剂，但是它们都需要进行单独的染色和脱色过程，步骤繁琐，操作要求高，且银染稍有不慎会造成很深的背景，影响结果的清晰度，最严重的是，有的银染胶脱色困难；考马斯亮蓝染色虽然染色背景低，但灵敏度低且消耗时间长。
发明内容
本发明所要解决的技术问题在于提供一种稳定性好、固色性强的荧光蛋白质染色剂，并为该染色剂提供一种制备方法和应用。
解决上述技术问题所采用的技术方案是该荧光蛋白质染色剂的结构式如下：
上述荧光蛋白染色剂的制备方法如下：
1、在0℃、搅拌条件下，将干燥的环己酮和浓硫酸混合，然后加入4-二乙氨基酮酸，待4-二乙氨基酮酸完全溶解后，升温至85～95℃，恒温反应1.5～2小时，冷却至常温，将反应液倒入冰中，再加入质量分数为70％的高氯酸水溶液，过滤并用冰水洗涤，所得固体真空干燥。
2、在0℃、搅拌条件下，将POCl3和N,N-二甲基甲酰胺按体积比为1:2.5～3.5混合；将步骤1真空干燥后的固体完全溶解于N,N-二甲基甲酰胺后，滴加到N,N-二甲基甲酰胺和POCl3的混合液中，滴加完后升温至85～95℃，恒温反应3～4小时，冷却至常温，将反应液倒入蒸馏水中，分离纯化产物，得到荧光蛋白染色剂。
上述步骤1中，优选4-二乙氨基酮酸与环己酮的摩尔比为1:1.5～2.5，干燥的环己酮和浓硫酸的体积比为1:10～15。
上述步骤2中，优选步骤1真空干燥后的固体与POCl3的摩尔比为1:1～1.5。
上述荧光蛋白染色剂在蛋白质染色中的用途，染色方法为常规蛋白质染色方法。
本发明的有益效果如下：
1、本发明染色剂的水溶液具有强烈的荧光，且该染色剂含有醛基(—CHO)，能与蛋白质上的氨基(—NH2)反应，形成共价键，利于在染色时与客体产生相互作用，染色牢固，不易脱色，提高了染色与荧光检测效果，提高了灵敏度，有利于荧光或裸眼识别与检测。同时，本发明染色剂具有色泽鲜艳、色谱齐全、优异耐洗牢度、价格相对便宜、应用工艺简便的特点。
2、本发明染色剂可以在蛋白质的预处理过程中与蛋白质结合进行染色，然后进行SDS-PAGE分离，分离后即可直接进行蛋白质的分析与检测，与传统的考马斯亮蓝及银染方法相比较，SDS-PAGE分离后不需要进行单独的染色和脱色过程，具有使用方便、操作步骤少、节约时间、灵敏度高的优点。
附图说明
图1是本发明荧光蛋白质染色剂的荧光光谱图。
图2是牛血清白蛋白的染色图。
图3是溶菌酶的染色图。
具体实施方式
下面结合附图和实施例对本发明进一步详细说明，但本发明的保护范围不仅限于这些实施例。
实施例1
1、将35mL质量分数为98％的浓硫酸加入到干燥的圆底烧瓶中，再向圆底烧瓶中逐滴加入3.3mL(31.85mmol)干燥的环己酮，并将其冷却到0℃，然后在剧烈搅拌的情况下，向圆底烧瓶中分批加入总质量为5.0136g(16mmol)的4-二乙氨基酮酸，待4-二乙氨基酮酸完全溶解后，在90℃水浴锅中加热反应1.5小时，反应完后反应液颜色呈黑红色，将反应液冷却至常温后倾入到150g冰中，再加入3.5mL质量分数为70％的高氯酸水溶液，过滤并且用100mL冰水洗涤，所得固体在真空烘箱中45℃烘干。所得干燥产物的MS(ESI+)m/z:实测值376.1909[M+H]+，理论值[C24H26NO3]+:376.1907。
2、将1.5mL干燥的N,N-二甲基甲酰胺(DMF)加入到圆底烧瓶中，在冰水浴中冷却到0℃，在剧烈搅拌的情况下，将0.56mL(6mmol)POCl3逐滴缓慢加到圆底烧瓶中；将1.875g(5mmol)步骤1真空干燥后的固体用10mL DMF完全溶解后，缓慢加入到DMF和POCl3的混合液中，在90℃水浴锅中加热反应3小时，此时反应液颜色为红棕色；将反应液冷却至常温后倾入到水中，用二氯甲烷萃取并旋蒸后，在真空烘箱中45℃烘干，用石油醚与乙酸乙酯、甲醇的体积比为9:3:0.4的混合液为展开剂进行柱色谱分离提纯，得到荧光蛋白染色剂0.8859g，其产率为47.3％。
所得产物的结构表征数据为：MS(ESI+)m/z:实测值404.1858[M+H]+,理论值[C25H25NO4]+404.1856；1H NMR(400MHz,CDCl3,d/ppm)δ:10.50-10.17(m,1H),7.73-7.32(m,2H),7.30-7.12(m,1H),6.56(d,J＝9.3Hz,1H),6.35(d,J＝3.0Hz,1H),6.26(dd,J＝9.2,2.8Hz,1H),3.81-3.12(m,4H),2.61-1.36(m,5H),1.26(dd,J＝20.4,12.8Hz,3H),1.16(t,J＝7.4Hz,3H)。
将所得染色剂完全溶解于蒸馏水中，采用RF-5301型荧光分光光度计进行表征，结果见图1。由图可见，此染色剂的激发波长为476nm，发射波长为564nm。
实施例2
实施例1制备的荧光蛋白质染色剂在牛血清白蛋白染色中的用途，具体方法如下：
样品的预处理：将荧光蛋白质染色剂完全溶解无水乙醇中，再加入蒸馏水，配制成1mg/mL的荧光蛋白质染色剂溶液；将50μL 1mg/mL的荧光蛋白质染色剂溶液和20μL 1mg/mL牛血清白蛋白水溶液加入25μL pH 8.0的Tris-HCl缓冲液中，并加入10μL甘油，充分混合后，在57℃下加热1小时，在3000rpm的转速下离心10min。
电泳分离：取15μL离心后的上清液加入聚丙烯酰胺凝胶(分离胶7.5％，浓缩胶4％)的泳道中，下槽中加入1000mL电泳缓冲液(3.0g Tris、14.4g甘氨酸、剩余为蒸馏水)，在浓缩胶部分施加100V的恒压，当鲜红色的条带跑至分离胶部分时，电压升至150V，直到条带跑至凝胶下边缘1cm处时，停止电泳，在凝胶成像仪上拍照，结果见图2。由图可见，本发明荧光蛋白质染色剂能够对牛血清白蛋白进行染色。
实施例3
实施例1制备的荧光蛋白质染色剂在溶菌酶染色中的用途，具体方法如下：
样品的预处理：将荧光蛋白质染色剂完全溶解无水乙醇中，再加入蒸馏水，配制成1mg/mL的荧光蛋白质染色剂溶液；将50μL 1mg/mL的荧光蛋白质染色剂溶液和20μL 1mg/mL溶菌酶水溶液加入25μL pH 8.0的Tris-HCl缓冲液中，并加入10μL甘油，充分混合后，在57℃下加热1小时，使溶菌酶充分变性，在3000rpm的转速下离心10min。
电泳分离：取15μL离心后的上清液加入聚丙烯酰胺凝胶(分离胶16.5％，夹层胶10％，浓缩胶4％)的泳道中，下槽中加入1000mL电泳缓冲液(3.0g Tris、14.4g甘氨酸、剩余为蒸馏水)，在温度为4℃、电流为25mA的条件下进行电泳，直到条带跑至凝胶下边缘1cm处时，停止电泳，在凝胶成像仪上拍照，结果见图3。由图可见，本发明荧光蛋白质染色剂能够对溶菌酶进行染色。
Claims (6)
1.一种荧光蛋白质染色剂，其特征在于该蛋白质染色剂的结构式如下所示：
2.一种权利要求1所述的荧光蛋白质染色剂的制备方法，其特征在于它由下述步骤组成：
(1)在0℃、搅拌条件下，将干燥的环己酮和浓硫酸混合，然后加入4-二乙氨基酮酸，待4-二乙氨基酮酸完全溶解后，升温至85～95℃，恒温反应1.5～2小时，冷却至常温，将反应液倒入冰中，再加入质量分数为70％的高氯酸水溶液，过滤并用冰水洗涤，所得固体真空干燥；
(2)在0℃、搅拌条件下，将POCl3和N,N-二甲基甲酰胺按体积比为1:2.5～3.5混合；将步骤(1)真空干燥后的固体完全溶解于N,N-二甲基甲酰胺后，滴加到N,N-二甲基甲酰胺和POCl3的混合液中，滴加完后升温至85～95℃，恒温反应3～4小时，冷却至常温，将反应液倒入蒸馏水中，分离纯化产物，得到荧光蛋白染色剂。
3.根据权利要求2所述的荧光蛋白质染色剂的制备方法，其特征在于：在步骤(1)中，所述4-二乙氨基酮酸与环己酮的摩尔比为1:1.5～2.5。
4.根据权利要求2或3所述的荧光蛋白质染色剂的制备方法，其特征在于：在步骤(1)中，所述干燥的环己酮和浓硫酸的体积比为1:10～15。
5.根据权利要求2所述的荧光蛋白质染色剂的制备方法，其特征在于：在步骤(2)中，所述步骤(1)真空干燥后的固体与POCl3的摩尔比为1:1～1.5。
6.权利要求1所述的荧光蛋白质染色剂在蛋白质染色中的用途。
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