查看完整版本请点击这里：
【求助】为什么我洗不下来的His-TAG的蛋白呢？？
我表达的蛋白大小在31 kDa左右。在N端有6个His.我在NATIVE 的条件下，用的binding buffer 50mM NaH2PO4, 500 mM NaCl, Washing buffer 是相同条件的binding buffer 加 20 mM imidazole. Elouting buffer 是相同条件的binding buffer加上250 mM imidazole. pH 值都是 8.0【求助】为什么我洗不下来的His-TAG的蛋白呢？？
用的是QIAGEN的 镍柱。但是最后发现在washing buffer, eluting buffer里面都没有一点蛋白。蛋白还都在裂解液里面。
我在混合binding buffer和蛋白裂解液的时候，是在4°下摇晃一小时。（本想减少其他蛋白的非特异性结合）
请问我纯化不下蛋白的原因是不是出在温度过低？还是我的buffer条件应该调整？？谢谢大虾们的指教！！
查看完整版本请点击这里：
【求助】为什么我洗不下来的His-TAG的蛋白呢？？
【求助】为什么我洗不下来的His-TAG的蛋白呢？？
最新回复
wood533 (2013-12-13 17:39:34)
条件不明：
1、蛋白裂解液的成分？
2、QIAGEN 镍柱的名称？
3、镍柱是如何进行预处理的？
4、胶用量？蛋白质样品用量？
5、SDS-PAGE图？
fsdd817 (2013-12-13 17:40:40)
QUOTE:
蛋白裂解液成分不明，是“PRO-PREP” Protein Extraction Solution,产品说明书只是说有5种蛋白酶抑制剂，其他成分无。QIAGEN Ni-NTA Agarose 25 ml 装。
因为只想纯化 2ml 的裂解蛋白，所以只取 1 ml 混合 NI-NTA液，置于伯乐0.8x4的柱子中，室温精致15分钟，移除上清。然后用 DDW 清洗两次，移除上清。
36448333.jpg
fsdd817 (2013-12-13 17:41:06)
左边是 Marker,左起是，elution 1,2,3,4,5.washing 1,2,3,最后一行是Flow throw.
就是目的蛋白在flow throw 里，而不在洗脱液中
wood533 (2013-12-13 17:42:02)
3、镍柱是如何进行预处理的？
6、样品来源？为什么要用这种裂解液？
wood533 (2013-12-13 17:42:29)
PRO-PREP Protein Extraction Solution (C/T)
Description
By using PRO-PREP(TM), proteins can be simply extracted from all kinds of cells and tissues. The kit contains 5 kinds of protease inhibitors so it is possible to extract very highly purified proteins . Usually detergent used in protein extraction consists of both hydrophobic tails an amphiphilic molecule and hydrophilic head. The two parts are joined to form a micelle, that is, solubilized protein forming a lipid-detergent mixed micelle and transmembrane protein forming a protein-lipid-detergent complex. The extent of micelle formation is termed as CMC (critical micelle concentration), which is important for high efficiency as well as high purity of protein extraction. CMC is influenced by pH, temperature, ionic strength, multivalent ions of organic solvents, purity of detergent, and so on. Depending on its ionic characteristics, a detergent can be categorized as ionic detergent, non-ionic detergent, and Zwitterionic detergent. Ionic detergent can be further classified into either cationic detergents : SDS, LiDS and DOC and anionic detergent. Thus these are highly denaturant which have a specific property to isolate protein as a monomeric form and so often used in Western blot analysis and measurement of molecular weight. Also non-ionic detergent such as Triton X-100 are less protein denaturant and often employed in protein-protein interaction.Zwitterionic detergent such as CHAPS have both negative and positive charge head at the same time, more effective in protein-protein interaction than non-ionic detergent and its extent of protein denaturation is less than that of ionic detergent. It is very important to select the optimal buffer and detergent when extracting proteins.
Characteristics
When extracting proteins from cells or tissues, one doesn’t necessarily apply appendix treatment.
- Able to minimize protein extraction time into 20-30 minutes.
- Protein stabilization buffer can make protein stable.
- There is no protein degradation due to freezing or thawing since there is no freezing at -20℃ reservation.
- Extracted proteins are stable for more than 6 months when kept in –20℃.
- There is no absorbable error because there is no absorbable hindrances of PRO-PREP solution when measuring protein concentration.
- Very useful for protein separation in Western blot analysis because ionic detergent turns protein into monomers.
- Very useful for protein molecular weight analysis because denature protein into a monomers.
- Protein degradation is minimized by adding commonly used protease inhibitor and doesn’t necessarily need to prepare protease inhibitor.
Protease Inhibitors in PRO-PREP Solution
PMSF
(phenylmethylsulfonyl fluoride)
- inhibits serine protease and thio protease
- added in a working concentration of 1.0mM (174ug/ml)
wood533 (2013-12-13 17:43:08)
(ethylendiamine tetraacetic acid)
- inhibits metaloprotease
- added in a working concentration of 1.0mM
Pepstatin A
- inhibits acid protease
- added in a working concentration of 1uM (0.7ug/ml)
Leupeptin
- inhibit serine protease
- added in a working concentration of 1uM (0.5ug/ml)
Aprotinin
- inhibit serine and thiol protease
- added in a working concentration of 0.1uM (2.0ug/ml)
Applications
Western blot assay, Protein-protein interaction, Immunoprecipitation, etc
Kit Contents
PRO-PREP(TM) Solution 100㎖
Storage
-20C (stable at least 1 year)
是这个东东吗？
59316371.snap.jpg
fsdd817 (2013-12-13 17:44:52)
QUOTE:
对，就是那个裂解液。因为实验室之前一直用这个，所以我就沿用了。柱子没有什么别的特别预处理了，就是先把树脂挂柱，然后用DDW洗一次。就加 3 ml binding buffer 翻覆颠倒混匀，然后静置移除上清。最后加 2 ml 裂解液，在4度条件下摇匀1小时。
wood533 (2013-12-13 17:46:12)
EDTA
(ethylendiamine tetraacetic acid)
- inhibits metaloprotease
- added in a working concentration of 1.0mM
裂解液中有EDTA1.0mM ，造成镍柱无法吸附。
建议不要用所谓的裂解液，蛋白酶抑制剂对纯化来说，在绝大多数时候是没有什么意义的。就用普通的镍柱平衡buffer，超声破菌。
fsdd817 (2013-12-13 17:46:43)
QUOTE:
谢谢提醒，我也自己再多考虑，必进换新的蛋白提取液，跟老板得有个强有力的理由。fsdd817 (2013-12-13 17:51:07)
实验室有人一直在用上面提到的 PRO-PREP 裂解液，说用的挺好。没见1.0mM 的EDTA对其有多大影响。所以我有延续使用了几次。
之前，eluting 之后没有目的蛋白，恐怕是buffer 加的太多，"invitrogen"的protocol里面提到。50 ml培养液，用8~10 ml 的elution buffer 洗脱。后来，有人说这个buffer太多，我就缩小的buffer 的用量，换作200微升x5 次。可以得到蛋白，浓度大概在1 mg/ml左右。
在这当中，除了buffer 用量过多，还存在其它问题吗，因为蛋白的浓度并不高？
fsdd817 (2013-12-13 17:53:13)
蛋白的浓度为什么总是相对很低呢？前两次的蛋白浓度A280 在0.4 左右。 Eluting 1,2,3,4,5 浓度渐减，但有时候会出现陡然增高，重复几次测试陡然增高的浓度样品，又有所回落。有人提醒是 incubation 的时间，一般在30分钟左右，但我 4度 15分钟，这有大影响嘛？
【求助】为什么我洗不下来的His-TAG的蛋白呢？？