您的位置 首页 技术频道

能不能成为好蛋白,生活环境很重要

和蛋白打了好些年的交到,也多少了解了些他们的脾气,他们有些时候确实敏感细腻,稍有招待不周,就集体罢工、组团报废。不论是纯化的或是混合的,总之让蛋白们高兴地活着是一件很重要的事,通常情况下,这就是说要让这些蛋白不聚结、能溶解而且有活性。那么我们就需要给蛋白营造一个好的生活环境,也就是一个适当的缓冲液(buffer)同模拟生物细胞内部的稳态。在选择一个适当的缓冲液前,有几点是需要考虑的:1)缓冲液的pH;2)缓冲液的种类;3)盐浓度;4)还原剂;5)稳定剂。

pH

一般情况下,蛋白是要待在pH7.4的环境下,目的是模拟细胞稳态以了解蛋白在细胞内的功能。如果你的蛋白在这个pH下是稳定的,那么pH7.4就是个不错的选择。如果他们不稳定,则需要调整缓冲液的pH。一般经验告诉我们,当pH=pI的时候,即pH就是这个蛋白等电位点的值得时候,蛋白不太容易溶解(不知道什么是pI的,自行百度吧>^<)。查pI值的快速方法就是使用ExPASy ProtParam 进行计算(http://web.expasy.org/protparam/)。

缓冲液种类

一旦决定了pH值,接下来第一件事就是选择一个你要用的缓冲液,一定要保证这个缓冲液在所选pH上能够有缓冲能力,所以所选缓冲液的pKa(酸度系数)最好能在所选pH值所代表的酸电离度的附近。第二件事就是确定缓冲液的浓度,一般浓度是50-100 mM,有时候缓冲液浓度不能太高,以免影响蛋白的活性。第三件事就是注意到一些蛋白和缓冲液的特性,比如,磷酸基团会影响到激酶的活性,所以在溶解激酶的时候,要避免使用带磷酸基团的缓冲液,或者要把磷酸基团透析出去;再比如,很多缓冲液的pH是和温度有很大关系的,有时候在常温下配的缓冲液达到了我们想要的pH,但我们把缓冲液保存在4度时或是蛋白反应要在37度,pH变化会很大,最有名的就是Tri 缓冲液,pH会很大程度上受到温度影响。

盐浓度

常用于各种蛋白缓冲液的盐自然是NaCl,使用盐的目的是让蛋白质更容易溶解在缓冲液里,同时模拟蛋白生活的生理环境(physiological conditions of cells)。小编一般用150 mM NaCl在蛋白缓冲液中,但是根据情况的不同,盐浓度是可能会改变的。比如说不同阶段的蛋白纯化步骤,就像用离子交换柱纯化特定蛋白的时候,我们总归是先用低盐的缓冲液把蛋白输送进虑柱,而使用高盐缓冲液滤出蛋白。升高盐浓度是十分容易的事情,即配比缓冲液时多溶些盐;降低盐浓度,则通常使用透析的方法。

还原剂

如果目标蛋白含有半胱氨酸(cysteine)这样的氨基酸,那么防止-SH基团被氧化而导致的蛋白聚合是十分必要的。那么我们的缓冲液中要按情况添加一定浓度的还原剂。常见的还原剂有DTT(Dithiothreitol)、TCEP(tris(2-carboxyethyl)phosphine)或者2-mercaptpethanol(BME)。TCEP(tris(2-carboxyethyl)phosphine)是这三种中最稳定也是最贵的。实验室里常见的是DTT和BME这两种,小编一般会把这些还原剂的浓度保持在5-10 mM左右(基本上是要比蛋白浓度要高的)。由于DTT和BME,小编一般是配置无还原剂的缓冲液,等到要用的当天,把储存在冰箱里的DTT或BME按一定比例配比到缓冲液里。特别要注意的是,在某些蛋白纯化的时候,一定要查看所用虑柱是否可以接受这些还原剂,比如说镍柱(His-tag亲和纯化)就会被高浓度的还原剂还原,虑柱颜色由蓝变绿,导致虑柱失活。

稳定剂

一般来说,除了上面提到的四种元素外,我们还可以向蛋白缓冲液中加很多东西,去帮助提高蛋白这种缓冲液中的溶解度和在稳定性,小编就统称这些东西为稳定剂。稳定剂的种类、多少等等都要依情况而定。举几个例子,加入BSA(bovine serum albumin)有时候确实可以稳定住其他蛋白;有时候,通过加入甘油(glycerol)或PEG(polyethylene glycol)增加缓冲液粘稠度,确实可以增加蛋白的稳定性;有时候甚至可以加入一些清洁剂/去污剂(e.g. Triton 或者Tween),也会增加稳定性。

如何配置适合的蛋白缓冲液,需要我们在实验室不断尝试,并且依照情况灵活改变的。有什么关于配置蛋白缓冲液的想法,欢迎留言讨论哦!

文中出现的一些物质的结构

2016032801

图片来自网络,内容由BioEngX原创,转载请注明!


扫描下方二维码关注BioEngX官方微信公众平台
qrcode_for_gh_1d4074a25cf9_258

作者: 卢阳

伦敦大学学院博士,研究方向为生物催化和细胞色素P450开发;BioEngX创始人之一。知乎主页:簏岩。



发表回复

您的电子邮箱地址不会被公开。 必填项已用*标注

评论列表(87)

  1. An impressive share, I simply given this onto a colleague who was doing somewhat analysis on this. And he in truth bought me breakfast as a result of I found it for him.. smile. So let me reword that: Thnx for the treat! But yeah Thnkx for spending the time to discuss this, I really feel strongly about it and love reading more on this topic. If possible, as you turn into expertise, would you thoughts updating your weblog with extra details? It’s extremely useful for me. Big thumb up for this blog put up!

    https://youtu.be/8_QSy0Zy1MY

  2. What i don’t realize is if truth be told how you’re now not actually a lot more neatly-preferred than you might be right now. You are very intelligent. You know therefore considerably when it comes to this topic, made me individually believe it from a lot of various angles. Its like men and women are not fascinated until it?¦s one thing to do with Lady gaga! Your own stuffs nice. Always take care of it up!

    https://youtu.be/xGsvEIoZBtY

  3. A powerful share, I just given this onto a colleague who was doing a bit of evaluation on this. And he the truth is bought me breakfast because I found it for him.. smile. So let me reword that: Thnx for the deal with! However yeah Thnkx for spending the time to debate this, I feel strongly about it and love studying more on this topic. If potential, as you change into experience, would you thoughts updating your weblog with more particulars? It is extremely useful for me. Huge thumb up for this blog publish!

    https://youtu.be/F_CSbrHz8IQ

  4. Hi there! This post couldn’t be written any better! Reading through this post reminds me of my previous room mate! He always kept talking about this. I will forward this article to him. Pretty sure he will have a good read. Thank you for sharing!

    https://youtu.be/9VtJwFIckBY

  5. I think other web site proprietors should take this site as an model, very clean and fantastic user genial style and design, as well as the content. You are an expert in this topic!

    https://youtu.be/se3fAAfYujs

  6. Whats Going down i am new to this, I stumbled upon this I’ve discovered It positively helpful and it has aided me out loads. I am hoping to give a contribution & aid different customers like its aided me. Great job.

    https://www.revtut.com/about/

  7. You could certainly see your expertise in the work you write. The sector hopes for even more passionate writers such as you who aren’t afraid to mention how they believe. Always follow your heart. “Faith in the ability of a leader is of slight service unless it be united with faith in his justice.” by George Goethals.

    https://roketslotlow.com

联系我们

联系我们

(44)07934433023

在线咨询: QQ交谈

邮箱: info@bioengx.org

关注微信
微信扫一扫关注我们

微信扫一扫关注我们

关注微博
返回顶部