CN111474032A - 一种高灵敏度快速sds-page蛋白胶染色液 - Google Patents
一种高灵敏度快速sds-page蛋白胶染色液 Download PDFInfo
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- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 32
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 32
- 239000012192 staining solution Substances 0.000 title claims abstract description 31
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 title claims abstract description 20
- 239000003292 glue Substances 0.000 title claims abstract description 15
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 13
- 229920001353 Dextrin Polymers 0.000 claims abstract description 9
- 239000004375 Dextrin Substances 0.000 claims abstract description 9
- 235000019425 dextrin Nutrition 0.000 claims abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 7
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 7
- 239000008213 purified water Substances 0.000 claims abstract description 5
- 239000000463 material Substances 0.000 claims abstract description 3
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 claims abstract 2
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 238000003756 stirring Methods 0.000 claims 1
- 238000004043 dyeing Methods 0.000 abstract description 19
- 239000007788 liquid Substances 0.000 abstract description 7
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 238000002360 preparation method Methods 0.000 abstract description 3
- 231100000053 low toxicity Toxicity 0.000 abstract description 2
- 238000010186 staining Methods 0.000 description 12
- RWVGQQGBQSJDQV-UHFFFAOYSA-M sodium;3-[[4-[(e)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-n-ethyl-3-methylanilino]methyl]benzenesulfonate Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=C1 RWVGQQGBQSJDQV-UHFFFAOYSA-M 0.000 description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 8
- 229940098773 bovine serum albumin Drugs 0.000 description 8
- 239000000243 solution Substances 0.000 description 5
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 3
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 238000004042 decolorization Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 239000002085 irritant Substances 0.000 description 1
- 231100000021 irritant Toxicity 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
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- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Abstract
本发明的一种高灵敏度快速SDS‑PAGE蛋白胶染色液,每500ml染色液由以下份量的材料制备而成:糊精5g~10g;考马斯亮蓝G‑250 25mg~75mg;无水乙醇40ml~60ml;磷酸40ml~60ml;余量为纯化水。本发明的染色液毒性小,在配制和使用过程中能够有效减少对操作人员的危害,以及避免对实验环境带来污染;操作过程十分简单,仅需将蛋白胶浸泡在染色液中,然后置于摇床上染色即可看到条带,无需进行固定和脱色处理;灵敏度高,染色20分钟可分辨凝胶中30ng的蛋白条带;反应迅速,蛋白量达6μg时,仅需1分钟就可以显出条带。
Description
技术领域
本发明的技术方案涉及分子生物学实验领域,具体涉及一种SDS-PAGE蛋白胶染色液的配制方法。
背景技术
聚丙烯酰氨凝胶电泳是一种在分子生物学实验中应用十分广泛的技术手段,其中蛋白胶的显色是十分关键的一步,传统的显色方式主要分为染色和脱色两步,在这一过程中所使用到的染色液和脱色液毒性较大,在操作过程中极易造成刺激性气体的挥发,给实验人员带来危害,而且需要反复进行脱色,过程十分繁琐。近几年新发展起来的一种蛋白胶染色仪,可以做到对蛋白胶的快速染色,操作简便,但是在使用过程中成本十分昂贵,大多数实验室都无法承受,故而淡出市场。
为了寻找一种操作过程简单易行且毒性低的染色液,本发明配制了一种无需脱色的蛋白胶染色液,并且通过应用发现该染色液染出的条带非常清晰,所需时间也很短。
发明内容
本发明所要解决的技术问题是:提供一种无需脱色、操作简单易行、灵敏度高、反应迅速且毒性低的蛋白胶染色液。
本发明解决该技术问题所采用的技术方案如下:
一种高灵敏度快速蛋白胶染色液,由以下份量的材料制备而成:考马斯亮蓝G-2500.005%-0.015%,无水乙醇8%-12%,磷酸8%-12%,糊精1%-2%。
该染色液具有以下优点:
毒性小,在配制和使用过程中能够有效减少对操作人员的危害,以及避免对实验环境带来污染;操作过程十分简单,仅需将蛋白胶浸泡在染色液中,然后置于摇床上染色即可看到条带,无需进行固定和脱色处理;灵敏度高,染色20分钟可分辨凝胶中30ng的蛋白条带;反应迅速,蛋白量达6μg时,仅需1分钟就可以显出条带。
附图说明
图1:本发明染色液对蛋白SDS-PAGE染色效果图;
图2:传统CBB G-250染色液对蛋白SDS-PAGE染色效果图。
其中:M:蛋白分子量标准;1-6分别对应上样量6μg、3μg、0.6μg、0.3μg、60ng以及30ng的牛血清白蛋白。
图3:本发明实施例2配制的染色液对蛋白SDS-PAGE染色效果图;
图4:本发明实施例3配制的染色液对蛋白SDS-PAGE染色效果图;
其中:M:蛋白分子量标准;1-3分别对应上样量6μg、0.6μg、60ng的牛血清白蛋白。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明,应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1
本发明实施例公开了一种高灵敏度快速蛋白胶染色液,包括如下成分:考马斯亮蓝G-25050mg、无水乙醇50ml、磷酸50ml、糊精7.5g、纯化水400ml。
同时采用本发明和传统CBB G-250染色液分别对两块上样量相同的蛋白SDS-PAGE进行染色,比较染色结果以及灵敏度。
蛋白样品上样:通过聚丙烯酰氨凝胶电泳,以牛血清白蛋白(BSA)作为标样,配制1mg/ml、0.5mg/ml、0.1mg/ml、0.05mg/ml、0.01mg/ml、0.005mg/ml的BSA溶液。上样体积均为6μl。最终上样量分别是6μg、3μg、0.6μg、0.3μg、60ng、30ng。
蛋白聚丙烯酰胺凝胶的染色过程:在避光的塑料器皿中分别倒入本发明染色液以及CBB G-250染色液各30ml,将蛋白胶分别放入染色液中,然后置于摇床上染色20分钟,蛋白胶取出用水冲洗两遍后浸泡在水中。染色液进行回收重复利用。
实验结果:两种染色液对蛋白SDS-PAGE的染色效果图如附图所示(图1:本发明染色液的染色结果,图2:CBB G-250染色液的染色结果),本发明染色液染色的蛋白SDS-PAGE条带清晰,背景干净,在相同染色条件下,灵敏度优于传统CBB G-250染色液。
实施例2
本发明实施例提供的染色液,包括如下成分:考马斯亮蓝G-250 50mg、无水乙醇50ml、磷酸50ml、糊精5g、纯化水400ml。
蛋白样品上样:通过聚丙烯酰氨凝胶电泳,以牛血清白蛋白(BSA)作为标样,配制1mg/ml、0.1mg/ml、0.01mg/ml的BSA溶液。上样体积均为6μl。最终上样量分别是6μg、0.6μg、60ng。
染色过程同实施例1。
实验结果:本发明染色液对蛋白SDS-PAGE的染色效果图如附图3所示,加入5g糊精的染色液染色的蛋白SDS-PAGE条带清晰,背景干净,灵敏度高。
实施例3
本发明实施例提供的染色液,包括如下成分:考马斯亮蓝G-250 50mg、无水乙醇50ml、磷酸50ml、糊精1g、纯化水400ml。
蛋白样品上样过程同实施例2
染色过程同实施例1。
实验结果:本发明染色液对蛋白SDS-PAGE的染色效果图如附图4所示,仅加入1g糊精的染色液染色的蛋白SDS-PAGE条带较弱,灵敏度相对较低。
Claims (2)
1.一种高灵敏度快速SDS-PAGE蛋白胶染色液,其特征在于,每500ml染色液由以下份量的材料制备而成:
糊精5g~10g;考马斯亮蓝G-250 25mg~75mg;无水乙醇40ml~60ml;磷酸40ml~60ml;余量为纯化水。
2.按照权利要求1所述的制作方法,其特征在于:
首先,将考马斯亮蓝G-250、无水乙醇、磷酸依次加入水中,搅拌20分钟,确保样品全部溶解。
然后,在上述样品中加入糊精,待样品完全溶解后置于4℃,闭光保存。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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CN101473228A (zh) * | 2006-04-28 | 2009-07-01 | 伊克斯派德恩有限公司 | 使用染料和糊精的蛋白质检测试剂和方法 |
CN104004382A (zh) * | 2014-05-20 | 2014-08-27 | 北京五康新兴科技有限公司 | 一种考马斯亮蓝染色液和染色方法 |
CN108871911A (zh) * | 2018-07-06 | 2018-11-23 | 辽宁钰航生物医疗科技有限公司 | 一种叶酸复合物及其制备方法与应用 |
CN109368808A (zh) * | 2018-12-14 | 2019-02-22 | 南开大学 | 一种折流式固定床载体处理畜禽粪污水的装置及应用 |
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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CN101473228A (zh) * | 2006-04-28 | 2009-07-01 | 伊克斯派德恩有限公司 | 使用染料和糊精的蛋白质检测试剂和方法 |
CN104004382A (zh) * | 2014-05-20 | 2014-08-27 | 北京五康新兴科技有限公司 | 一种考马斯亮蓝染色液和染色方法 |
CN108871911A (zh) * | 2018-07-06 | 2018-11-23 | 辽宁钰航生物医疗科技有限公司 | 一种叶酸复合物及其制备方法与应用 |
CN109368808A (zh) * | 2018-12-14 | 2019-02-22 | 南开大学 | 一种折流式固定床载体处理畜禽粪污水的装置及应用 |
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