风了时间

2012年4月9日，停下了追逐肖梅艳的脚步。
5月1日，到京山寻亲。
5月10-13，公司组织厦门旅游。
6月21日晚上-24日晚端午假期回家探亲。

千里寻母

2012年4月29日，我来到了湖北京山县，当晚在京山县城过夜，第二天早上坐汽车来到钱场镇，然后转车到龙泉，在包一个面包车进徐冲村，在徐冲村二组我见到了19年没有联系的外婆，想从她那里寻些母亲的信息，无奈，他们也不知道母亲的去向，据外婆猜测是姓严的人贩子将母亲骗走了，哎……寻母的计划就这样开始了，一切都没有那么容易，毕竟二十年了，人和事都不会太清晰了……道路漫长，希望母亲过得好。图为外婆及其两个孙子。

随清风吹走

2012年4月9日，在肖梅艳与其所谓现任男友给我发视频后，我决定放弃对她的追逐，我无法改变她喜欢的生活，所以也无法进入她的生活，祝福他们吧！

记录

08905

看看有没问题，没问题就发过去吧

她还是不信，说你想骂的时候就骂，想让人原谅你就原谅，慢慢来，千万不要着急

你加她QQ吧，她曾经说过你删了她，但不能说以前那些话，想要原谅就说原谅的话

对了，本来就是你错在先，你有什么资格发脾气，就是因为你脾气或者说你没控制自己的脾气才会有今天的结果

说明还是有机会的，呵呵

我再提醒你不要再说错话了

恩，她电脑拿下来了

不管以后怎样，你一定不能让小六知道我在帮你

08906

E你知道你在校内上的两句话带来多大影响，小六不是因为那个她会考虑原谅你的

小欣早上可能找你谈话，你要跟她道歉，因为你也伤害了她，只要道歉

E，好好表现哦，这步很重要

后来那句也不行，不要老是以这种方式自我释放行不行

嗨，事态严重性？都你自己想出来的吧

还有你以前对小欣说的话，也是要表达事态严重性，那些话只会让人更生气

08912

是不是要球拍，已经拿去了

09526

508

09618

严代才，能帮我找一个北美大学的课程体系吗

再给周皓芹找一个吧

严代才，你有微生物备考资料，没答案的那份没

有的话给我发一下，谢谢（笑）

谢谢，你那么帮我，有需要我帮忙，一定挺身而出啊

09618

？谢我什么

09622

不要什么事都让我知道，她会更不高兴的，她如果想让我知道，她自己会说的，只要不要太恶意，以后注意点就好

她没那么小气，如果有什么误会需要解释的话，解释一下，没有的话就不要说了

我不是他，所以觉得挺正常。她可能希望你能像普通朋友那样正常点吧

上次你怎么呼叫转移到她手机上

那你给她评的好友印象她删了吗？

那就没事啊，她就是说说吧，别这么紧张，太在乎就会不正常的，自己体会一下吧

总之没事，不要想了

09626

自习室的座位还要不的，一个女生占了你旁边那个座位

小学期自习室应该会被用作课程设计

那你去看看吧

不知道现在有人没

什么时候都行啊

09701

昨晚睡了，什么事？

你去配把钥匙吧，多方便啊

我还没起呢，小红起了，我最近很头疼，心理上很想好好学习，生理上却很懒，不知道为什么会这样

觉得很累，学习很枯燥，似乎有点排斥，起来吧起来吧

09723

昨晚那时候已经睡了，找我有事吗？

09725

怎么了？今天我是想找人帮我取一包裹

09726

不记得了-以后别再用酒精来伤害自己的身体

在寝室吗？能借我篮球吗

不用了，我下回晚上跟你借吧

我的技术还是不要在白天占别人的场地了

09727

桂林路市场，找到没啊

喔，跑那么远啊先去旁边的恒克隆看看有没

呵呵，你要做什么啊，这么着急买

晚上不行，明天早上行不啊，严教练，教我打球啊

那等你以后有时间吧

09809

篮球场那么多人还怎么打啊

09810

书拿回来了，现在拿给你，还是要什么时候给你再看看

明天早上还打不打球啊

说得怎么样？感觉她心情还不错

什么意思？

哎！你们是平起平坐了，忘了以前的不愉快，好好做朋友吧？

其实可能除了她我们都能理解你，你并没有落魄丫

希望真好了，早点睡，六点球场见咯

09811

我跟她说了，不过怎么还有我呢，你们俩的事我去干嘛丫

09812

没事了，刚刚小红和我找你打球呢

六点小篮球场

09813

上QQ

不着急，已经没事了，上网跟你说一下

09814

去的啦，我是说要是室外就不去

会展中心应该是室内，所以要去的啊

明天打球继续啊，六点五分没到给我打电话

没关系，我也腿疼

歇一天吧，明天民博会也不去了吗

09815听小红说你去了人才市场了，有什么收获吗？我们在会展中心逛了一个小时就回来了

噢，少喝点，教练！别抽烟！酒后抽烟，危害更大！你今天是不是玩得太累了，要不明天再休一天？

那你几点才能睡觉啊

明天还是休息了，我有事！

我没说假话，本来就有事，我团契八点有早祷得，我想打球不想参加，感觉自己在逃避！

不太应该

09816

我要买篮球了，我们打的那个多少钱的

张景方说你又要去当保险经理了？噢想做就去做吧

只是你以后还要去参加早会，还能早起训练我打篮球吗？

会不会太累了？

不在哦，我在逸夫楼

你这个经理还可以有特殊待遇丫

脸皮这么薄，怎么做保险啊呵呵

以后变成一个做保险全家都光荣的，况且你们做经理又不做业务员，就谈不上了

09817

恩那

你怎么没上qq啊

哦，晚安！

多睡会儿，别那么早起了！

噢呵呵

09818

噢，马上起！

09819

噢，好

再接个热水，马上出去

09821

不打紧，大篮球场

我们教室的座都被人占了，他们还把我们书撇前面了

我的座还剩一个，其他人都没了，

大家气疯了怎么办？

你想怎么样？

你们太不理智了，别再闹了！

过是过了，我不喜欢这样的处事方式

不过拿会座咱们班又能在块自习还是有点开心的

09822

已经用桌布占了，我把我的一些草稿纸放你抽屉里了

09823

你那下雨，我这肯定也下吧，你就别出来了

我起了

知道了

09824

你又这么早！

膝关节好疼怎么办

明天还能不能打？

多久能好啊？

现在一动就疼，今天早上打的时候我都没感觉啊，怎么就这样了呢

晚上我用你的方法试试吧，一定要快点好

她说不打了

09825

弯的时候还是疼，我要去买喷雾剂喷，想快点好

膝盖还是疼，明天还是不打了

以你的经验几天能好哇

我要睡了，知道就行，不用给我回了

09828

明天我有事儿，看不到你们打篮球了好好玩，输赢不重要，玩得漂亮，玩得开心就好

09831

这几天忙吗？明天早上去打球好不好？

呵呵，我去了噢

去打球了，结果回来没带钥匙大家都没起被锁外面了

09901

马上下去

09902

睡迟了，马上起！

09903

你没在上网睡了吗？

明天不能打球了

09904

恩拉，以后早上6点半我要和一个学妹学习圣经啊，我们很早以前也一直做过的，后来停了一段时间，晚上学妹提出要重新开始学了

我们还能有别

09905

你在自习室？还挂着QQ的

你请小六吃饭没啊

晚上吗？估计我在睡觉，都不知道她出去过了

噢，晚上应该不去或者会完点去，你有事？

我是很不想学啊，

背了几个月了才背十几页还没完全记住

我想能保研还是保吧，好没用哦

我没信心一定保的，等成绩出来吧

他只要一个吗？我指的不能保湿任何情况下，反正我也摇摆不定的，没人要我就算了，到时候硬着头皮考吧

反正都是我们班的吧，有人想保他的酒保他的吧，我看看能不能保食品的

09906

大一军训，明天能去小篮球场了

09908 01:14

我们通宵了，早上不打球了呵呵

在外面呢

正要回去了

09910

在

哦，知道了

09911

你决定不保了吗？

保研就根据这个排名？

09912

你想清楚了吗？

现在我们这个专业工作不好找，你要是能保上农机肯定比现在好啊，你就试试吧

你去找老师吧，如果农机不行的，那就别保了行不

第六不是挺好的嘛，王刚没有比第六好吧，不也保了，试试你也没有损失

还有你做过那个实验，男生这个也是优势

09914

出来没？今天不打了

09915

好，今天干嘛去了，从早上开始就有事

噢，图画完了吗

09916

明天我要休息！呵呵

09918

明天我要睡觉！不打球！！

09923

我的电脑有问题了，你跟我说什么了吗？

慧慧也跟我们玩

09925

去。但是我还在外面呢，回去再打好，你多打一会儿，回去找你

刚到换个衣服就去

09929

明天跟老师说纠正一下排名的问题，再问一下第二名外保资格是我还是阳，拜托你了

091002

我查了差不多中午时免费的，我们十一点去西门坐车吧

还有谁想去也一起去吧，自费就行呵呵

去红旗街吧

网上说那好

091015

没有，打球是不是？

行，等我跟师哥说一声

去几餐啊

091019

思想表现，学业、外语，科研每个一句话就行

你走了？为什么

你怎么啦

091020

15948713226

091023

你还在篮球场吗？

我还在实验室，你会打到几点呀

噢，下回我有时间找你吧

091029

帮我把昨日的日程填上，上午：一餐一楼，东师遗传所，教务处 下午：二餐一楼，东师遗传所，寝室 晚上：寝室

昨天的信息填了吗？我发烧了，很严重

等一下去医院，39度，不知道是不是温度计坏了

中午我让阿梅收拾东西你给送一下

我在里面定晚餐了，你就别过来了，这里空气也不好，

要是出什么事怎么办呀退烧了

点了，我一两天应该就能出去，太感谢你了

能的拉，别来了让我安心住这吧，给我发发短信就行，

如果真有需要会告诉你的，有你这个同学我太幸福了呵呵

恩我会的呀，放心吧

恩走了，又来了一个

拜托噢，说了别来了啊…真的别来了，这里都有我需要的话会告诉你的

魔方我就是对着说明书学的，你也可以的

呵呵，那你看视频吧，我怕我讲不明白，我听力理解不怎么好，阅读比较好，所以大家都在跟视频学，我只能看说明书了

我学会了就是想要熟练一下啦，再次告诉你别来了，今天出去的那女生说她进来之前还没咳嗽，来了之后反而咳嗽了，说得我都害怕了

对了，之前我们办过四年的保险给我们保险单了吗

早餐里面也有，医生刚刚在说呢，进来医院可能把病毒带出去了，你不为自己想也为同学想啊，北航大一男生都牺牲了，今天你都来两次就挺冒险的了

恩，有什么事一定找你的，基本上应该没事，烧都退了，医院不好，没病都可能在里面变得有病了，要不是怕室友她们担心，我也不会来医院的

我肯定没事的，说这么多是让你真的别来了，不能来啊，你来一次我就担心一次

091030

已经定了，一会儿应该会送来

昨天靠窗睡有点冷，后来掉了个就好了，睡得挺好，除了做了个恶梦外

定餐现在不用直接付钱，等我能出了再算，在里面也就不用花钱了

恩，昨晚就正常了事情办完了，我也回医院了

没，即将睡，怎么了

不知道明天什么时候能出来呀

今天出去过了，错过两次

按昨晚退烧开始计60小时，要后天早上吧，我明天问问，能不能让我明天出去

恩晚安

091031

我明天才能出去

晚上出去多穿点衣服啊，明天再说吧

不用了

晚上笔试的是哪家啊

091101

十点以后，小六来接我，你就不用麻烦了

091105

怎么不来上课呀

091113

大家都相处了差不多四年了，都知道每个人都是什么样，所以尽管做你自己，你就是你啊

091117

你们都不来了吗

091211

我跟少帅通电话了，男生那他通知就行

今天好像又让你麻烦了，是我失误了，下不为例！真诚地跟你道歉！

091217

耳机借到了，不用问张林了，谢谢啦，有人为我的事这么上心真好！

呵呵，那我借你借的吧

091221

取回来了吗？好的吧，还有一个戴头上的皇冠吧先放你那，晚上再拿

091222

先放你那，晚上从东师回来去你宿舍取吧

091226

明天我团契过圣诞节，你要不要来参加

100108

说出去的话就是泼出去的水，太伤人了！弄成这样应该是我没经验，没做好工作，只希望大家可以一起开开心

100111

梁彦龙第八章计算机那部分考吗

那梁老师的号有人知道吗

100118

再见了！一路顺风！

100206

在家好忙好忙，正在开同学哦

100208

1号回去，你呢

100209

早上好！在家我都睡得很早哦

100209

那好像不是一些吧，多喝点水吧，以后喝了酒吃点水果会比较好

喝酒要适可而止，喝多了有什么益处呢，真不知道是谁发明酒这东西

100217

你干嘛认为我停机呀？买到车票了吗？

100218

我买到1号的票呵，又是坐的

你太好了，到时候再联系哦

100227

做的吗？是不是很累呀，辛苦了

100301

K552

太早了，你别来噢

不可以，后悔告诉你车次了

对啊所以你别来，白来就不好了，我手机肯定会没电的

你找不到我，你去车站不睡觉等空气呀，拜托你别来了

那你在寝室等我吧，我寝室那我怕行李拿不过去好吧

是真家伙还是电脑啊

什么不放心啊，我长得这么安全，再说也不是第一次，如果是白天，我会让你来接的，情况不同啦，相信我可以的啦

回去一起玩噢

拔牙

武汉大学口腔医院沌口门诊部

2012.3.10

拔掉了那颗虫牙

Affinity Chromatography of Histag protein

Many biochemists purify their beloved proteins, whether at small or large scale and for different reasons (identification, enzymatic reactions, characterization etc.). There are several rules and tips that can facilitate the purification process and increase your chances to have a pure protein in your freezer at the end of the day.

Pack enough beads to capture your protein but not too much to capture any other host protein

Predict and plan

The first step is to perform some basic bioinformatics analysis. The most important ones are those that relate to the physio-chemical properties of your protein such as amino acid composition, extinction coefficient, secondary structure prediction and motif prediction (see here for a comprehensive list of bioinformatics tools). Another good prediction website worth mentioning is Xtalpred, which acts as a meta server for the most basic protein analysis.

Once you gathered some basic knowledge about the protein, you can now assess:

• Membrane or cytosolic
• Number of cystienes
• Predicted pI
• Expected extinction coefficient
• Motif or structural domain which necessitates the addition of ions or co-factors

All the above will affect your expression system approach and your subsequent purification process. Plan ahead when to do an expression tests (different hosts, different induction media, different temperature and induction times). Evaluate how much protein you should require and plan your up-scaling expression accordingly. Check to see that you have the required materials (DNAse, protease inhibitors, columns and FPLC reserved) and prepare your schedule (you should look into Labguru, a web-based laboratory management system, that can help you manage your experiments).

Expressing your protein – making the most of every ml

This is one of the crucial steps in a succesfull purification scheme – you can do 5 purification steps and still be left with a dirty prep or do just 2 steps and get 95% and higher purity level. It is THAT crucial. Getting protein highly expressed in E. Coli is not always trivial but with some initial screening you might struck a successful induction setting (see my earlier post on how to improve recombinant protein expression). What settings should be checked?

• Media – Don’t settle only for LB and IPTG induction; you should try theAutoinduction media, developed by William Studier, which can boost protein expression between 3-10 fold, depending on the protein.
• Temperatures & induction time – Check several temperature points: 18, 27, 30 and 37 degrees Celsius, while varying the induction time appropriately. Meaning, the lower the temperature the longer induction time (we routinely induce proteins at 20 or 27 degrees over weekend). Start with an overnight and modify accordingly.
• Strains – Try inducing with Rosetta, Codon+, Tuner and Rosetta-Gami strains.

All the above settings should be tested at small scale (i.e. 50ml culture) and once you found a successful combination go ahead and up scale. And when I talk about up scaling I am talking about a minimum of 4L culture or higher. You might be surprised at this volume scale, but it is better to get a bucket loads of protein and then throw away major chunks as you cut out dirty fractions. No problem to start with 200mg of protein and then be left with 10mg of super pure protein.

Breaking cells or cell lysis

Assuming most will purify a recombinant protein, your protein will be expected to contain an N or C-terminal tag.

• When breaking cells try to use five volumes of lysis buffer per one gram of cell pellet.
• I found it best to redissolve the frozen pellet with the aid of a stirrer – this way you can continue preparing for the coming purification while the pellet slowly dissolves into the lysis buffer
• If you’re not purifying a membrane protein you can add 0.02% Triton X-100 to aid with the lysis of the bacteria.
• If you’re using His tag, don’t forger to add 10-20 mM Imidzole to the buffer
• Use ice all the times! The sample should be at 4°C all the time.
• Pre-chill the French press in ice about 10 minutes before intial lysis. If you’re having many samples you might want to chill it again for 5-10 minutes per several samples to keep it cold.

Initial separation and affinity chromatography

• Centrifugation at 45,000 rpm for 30-45 minutes is sufficient for most proteins (you can live also with half of it using SS-34 rotor at 20,000 rpm for 2 hours).
• If using a dripping column, initially pack beads that will be sufficient to ~10mg protein/1L of induction. After the first purification it will be easier for you to adjust the amount of beads to get the best purification without getting too much junk.
• Remember that in most cases the affinity chromatography step will determine the fate of your sample in terms of purity levels. Do your best to calibrate this step to the optimal setting possible so you get the protein at the highest purity level. From my own experience a dirty sample post affinity will not improve tremendously after using other columns (again, in most cases).
• Perform extensive washes, especially if working with dripping columns, 20 times the amount of beads volume (20 CV), varying the amount of salts and Imidazole.
• Elution should be conducted at a relative small flow rate to get the sample concentrated at the end of this step; in certain cases you’d want to increase the flow rate to avoid high concentration elution in case the protein is prone for aggregation.
• Evaluate your sample purity through an SDS-PAGE analysis. In case you find it hard to identify your protein do a western blot for verification (blot directly from the coomassie-stained gel).
• Depending on the purity level required and the observed purity of the sample, assess whether you want to do a step of ion-exchange or size exclusion.
• In case you plan on doing a step of ion-exchange, you might consider eluting your protein with low salt concentration (maximum 50 mM ion strength) and continue directly with your post-affinity elution into the ion-exchange column.

Purify, purify and…purify more: tips for improving your protein purification capabilities – part 2

In the previous post I have focused mainly on preparing and performing the first steps in the purification of a recombinant protein. In this section I will discuss a bit further about Nickel affinity chromatography via prepack column, about gradient elution and give some advice on improving purification of dirty preps.

HisTrap Prepack column from GE

Nickel affinity column gradient elution – capturing and purifying

While batch binding and elution step can be performed on a faster time scale (hour to hour and half from start to finish including wash steps), using a prepack nickel affinity column (such as HisTrap from GE) can help ending your first chromatography with a higher level of purity. This is true for all samples but it is crucial when you find your nickel affinity purification relatively dirty.

Why not use the prepack column from the first place?

It depends on the amount of proteins your purifying at a single moment (in our lab we sometime purify two to four proteins in parallel) and of course, if your sample volume is large it will take more time to bind, wash and elute.

When I am using a prepack column with gradient I still get the protein dirty no matter how much wash steps I do – what can be done to improve the nickel affinity purification?

From my own experience, when you have a good overexpression (i.e. higher than 15mg/1L of induction) then you will have better purification because the dominant protein in the sample will be your protein. However, if you have a low expression from the first place then your protein will have to compete with other host proteins which have similar expression levels. In such cases you should first consider improving expression via Autoinduction media (Studier et al. 2005) which I found to increase the fold of protein expression by 3-10 fold, depending on the protein. See my previous note about increasing expression.

Assuming you’ve tried everything and your protein is still low expressing you can do the following combination strategies, separately or combined:

• Decrease beads volume – if using a prepack column, try loading the protein on a 1ml column and not a 5ml. Why do that? Well, let’s assume your protein is expressing at 5mg/1L of media and you induced 4L of culture, giving a total of 20mg of protein of interest. If you will load the lysate on a 1ml of prepack column, that can capture 20-30mg of tagged protein, between 67% to 90% of the column will be captured by your protein. On the other hand, if you load the 5ml prepack column, with a potential capacity of 200mg (!), all the rest of the host proteins will have plenty of room to bind to.
• Removal of periplasm – Another strategy is to do periplasm wash of the bacterial pellet before performing the bacterial cell lysis. That’s because the periplasm was shown to contain many proteins and enzymes that naturally bind divalent/metal ion. By removing these protein you lower the host protein’s that compete over binding to the beads.
• Guanidinium/Urea wash – This is a bit risky but it is worth the try. With this strategy cells are lysed and bound to the nickel column. Then a wash with 2M of Guanidinium or Urea is performed which usually removes most of the host proteins and afterwards a shallow gradient of decreasing amounts of Guanidinium/Urea brings back your protein to native state. Of course, you will need to check and see that your protein didn’t loss its fold and function (usually if the protein refolds improperly you will notice aggregates once you concentrate your protein). The advantage of this technique is the ability to get a highly pure protein within one chromatography step.

Purify, purify and…purify more: tips for improving your protein purification capabilities

After doing our best to purify our protein at the affinity chromatography step, we might decide we want an additional step of purification, which in many cases involves either an Ion-exchange or size exclusion chromatography. In this part I will focus on Ion-exchange chromatography and some tips on how to maximize your time and effort to get the most of this system.

Ion-exchange chromatography (IEC) binding step

Ion exchange chromatography is a powerful technique that is used on many cases at the middle of the purification process. I have discussed a method how to improve peak resolution by repetitive Ion-Exchange chromatography and today I would like to discuss general consideration when working with ion exchange columns.

Unlike affinity chromatography, the elution in IEC is conducted via increase in ion strength or gradual change in pH that may affect your protein behavior (structure and/or function). Since binding of the sample to the column should be at minimum salt interference and maximum surface charge, usually there will be a requirement to lower the salt concentration. If the previous step was a His tag Affinity chromatography, it will save time if the elution step will contain a minimal amount of salt (e.g., ~50mM NaCl) so there will be no need to exchange buffer. If you’re wondering about the effect of the Imidazole in the solution, it depends on the type of IEC column used. If you’re using a cationic exchanger or working under pH 7 you have no need to exchange the imidazole simply because the fact that imidazole has a pKa of ~7 for its first nitrogen, thus at higher pH the ring will not be charged and will not interfere with the binding of the protein to the matrix.

The beauty about IEC is the fact it is a capture method: you can load as much volume as you want as long as you don’t overload the column with protein mass. This means you can start from 50ml or even 500ml diluted solution assuming you load your sample though a buffer valve. One thing to remember, though, is that there are proteins which do not bind to the anionic or cationic (or both!) so make sure you start collection immediately upon injection. Even in case your protein was not bound to the column, the contaminants might bind to the column so it is important to step-elute the bound material and evaluate it against the flowthrough and the pre-injected sample.

IEC elution step

When considering the elution step, two primary factors should be evaluated: elution buffer composition and elution methodology. In regard to the elution buffer it is usually recommended to use high concentration of salt (1-2M). pH shifts is less common since proteins are sensitive to small changes to the buffer’s pH which might affect their structure. On the other hand, it should be considered that gradual increase in salt can shift the protein from one oligomeric form to another (i.e. monomer vs. dimer) depending on its inter-molecular surface characteristics. Exploring other salts than the common NaCl is recommended when the protein aggregates or not eluting from the column.

The most common elution methodology is gradual increase in eluting buffer concentration (commonly known as “B buffer” or %B). While requiring more time, this methodology is essentially “fail-proof” as long as the gradient is relatively shallow such as 50%/50 min or 50%/10 column volume (CV) at 1ml/min flow rate using a 5ml column. Essentially you’ll want to elute the protein across 10-20CV so you’ll have better separation among close peaks. The slow rate will generate a much more concentrated elution which can expedite downstream concentration step.

Usually it is recommended to do a pilot run on the IEC. Inject small amount of the sample so you won’t overload the column (depending on the amount at hand, try to aim at 5-10mg) and evaluate the separation profile established with the current elution buffer composition and gradient. It is essential to couple a SDS-PAGE analysis of selected peaks together with the chromatogram so you can evaluate the following parameters:

• Peak separation
• Purity of the selected fractions
• Amount of injected sample – should you increase/decrease the amount?

At this point you can decide on keeping or changing the current settings.

When analyzing the peaks, one should remember that proteins can bind to the matrix through differently charged surfaces, which means that the elution profile of a 95% protein can generate several elution peaks. That’s why it is crucial to perform an SDS-PAGE analysis of the major peaks to verify whether a certain elution contains the protein of interest or a contamination. One should also remember that in certain cases what looks like a monomer on the gel can well be a dimer or an oligomer which forms a new charged patch that doesn’t exist in the monomer state; in certain cases, like crystallography, sample heterogeneity at the oligomeric level can be fatal for successful crystallization effort.

Step or gradient?

Moving from the gradient methodology to the step elution is a bit more complicated than it seems, especially when trying to separate between closely related peaks. Lets take the following scenario on the right.

IEC Gradient elution

In this purification run the main peak is bordered by two relatively small peaks. This was done by using an anion exchange column; color codes are blue color stands for 280nm, red for 254mn and brown for the conductivity in mS. Even though I would not recommend a shift from gradient to step in this particular case, I will use it to explain the workflow to perform such a change. First you should determine the volume in which you will want to start the step elution according to the absorbance curve (in this case I chose the inflection point, red dash line). From this volume deduct the total volume of the column (in this case 5ml) + additional 10% overhead in case of measurement errors. An additional way to determine the exact volume is to use the “%concentration” curve and determine the difference in volume between the starting point of mixing buffer A and B and the point at which there is increase in conductivity. I have done so manually (without the %concentration function) and established the volume at ~115ml (blue dashed line) afterwhich I have deduced the conductivity value at this specific volume. This will be the first elution step that will remove the preceding contaminant peak. The same can be done to separate the main peak from the late contaminant peak. Once you figure this out, repeat the test run with this step elution and see if it fits with your needs.

寂寞周末

周六值班，女博士瞎指挥，周末还安排大量培养，你培养得在早在多还不是得等我周一慢慢来纯化，蛋白不是你想出想出就能出的。
周日宅吧，早上看了NBA，老鹰胜了不可一世的雷霆。
这几天特别想恋那个X姓女孩儿，我都消失第4天了，为何还不联系我呢，这是要我彻底死心呀。

开始消失计划

为了测试我在匠科妹妹心中的地位，我决定消失一段时间，已经将qq托给老妹儿管理，让她改了我的密码，等我问她要的时候她再告诉我。
肖梅艳，你不能让我白白的浪费这么长的时间哟！相伴你走人生是我的梦想！

那些年，Y追过的女孩（原版）

《XYZ的故事》
——那些年，Y追过的女孩
看了《那些年》，想起了一个故事。
那一年，XYZ还在上高中……
X是美女，温文尔雅，文笔犀利，擅长散文，文字落落大方……Y英俊潇洒，风度翩翩，爱音乐，爱学习，
但也愤世嫉俗……Z跟X很像……
这个故事从二年级开是吧。
XY是高二年级同学，X和Y碰巧还是同桌，X是Y的第一位女同桌…（此处省略500字）…后来Y喜欢上了X，
喜欢上了她的文静，喜欢上了的她的文字，还喜欢上了转笔头的神情，重要的是喜欢上了她……但Y没有向X
告白，一是因为校规严禁中学生恋爱，二是害怕……觉得旁边的这位女生就像神一样不可打搅，不可亵渎…
就这样，XY没有发生任何故事，甚至他们都很少说话。
升三年级，X和Y被分到不同的班级去了……初始的时候Y还去X的教室外面去偷偷的看看她……直到Y跟同
班的Z混熟悉，他发现Z跟X有惊人的相似处，高考前他向Z表达了爱意，可是Z拒绝了，Y非常痛苦，每天都会以
写诗来减轻痛苦……
高考后，X去了省城，Y去了北方的城市，Z留下来补习，后来Z考上了本市的大学……
在大学，Y尝试过去喜欢别人，但没有。时间真快，大学毕业了……Y再次向Z表达爱意，但这时候Z已经有
了男朋友了……Y这次没有想象中的那么痛苦。Y醒悟过来了，原来他忘记了他爱的不是Z，而是X，Z在他心里
一直就是X的一种替代……他决心放弃Z，将心向月。

那些年，Y追过的女孩（彬校对）

《XYZ的故事》
——那些年，Y追过的女孩
看了《那些年》，想起了一个故事。
那一年，XYZ还在上高中，叛逆而又肆无忌惮的年纪…

很多时候都对于X的样子总是觉得很模糊，仔细想来X是美女，总是很安静的坐在那里，如果没人扰乱她的思绪，或者会让你觉得她有自己的世界，那是属于她一个人的空间，安静而美好。或者还有的印象就是一身的书卷味儿吧，可能那和她很犀利的文笔是脱不开关系的，而其中又尤擅长散文，文字落落大方又不失细腻，

……Y么，平时总是故作一副深沉的样子，或者这是那个年纪的孩子的通性，明明就一依靠家里的小屁孩儿，却硬要装的很强大，明明就单纯的跟一张A4白纸差不多，却硬要让人觉得有属于自己的个性；好吧，其友B给过一个评价是：就一愤世嫉俗的单纯小愤青…凭着有几分脑子，成绩居然也还差强人意。

……而Z，跟X很像……
高二那年，XY成了同班同学，碰巧还成为了同桌，作为Y的第一位女同桌，成功的获得了Y全部的注意力，实际上X本来就是个很能吸引人注意的女孩儿，安静而美好的样子，微微带点儿书卷气的气质……慢慢地，Y发现她越来越多的的样子，各种表情的她…静静地坐在书桌前的样子、转笔头时专注的神情、低头走路的样子、甚至是高傲着神圣不可侵犯的样子、淡漠的样子…彼时在学习和校规两座大山的压迫下，Y始终不曾做点儿什么，甚至后来的后来有了流言说他一直喜欢着谁谁谁，除了继续沉默，Y还是什么都没有做，更是由于他觉得X是那么安静而美好，不容打扰、不可亵渎…就这样，XY没有发生任何故事，甚至他们都很少说话，也没有任何人洞悉到Y的这个秘密。

升三年级，X和Y被分到不同的班级去了……初始的时候Y还经常不经意的溜达到X的教室外面不经意的看几眼，甚至偶尔只能看见空空的座位，直到某一天，Y在自己的班上发现了另一个秘密，有一个人和X有着惊人的相似度，所以慢慢地刻意接近Z，直到混得很熟悉，慢慢地Y可以不用去偷偷地看X了，看着眼前的Z他觉得更能接近，虽然Z安静的样子很像X，却更容易接近，没有那么美好到让人觉得只能远远地看着，也许是这样吧，Y认为自己喜欢上了Z,然后追逐-告白-失败-失落。
高考后，X去了省城，Y去了北方的城市，Z留下来补习，再后来Z考上了本市的大学…然后Y继续在对Z自以为是的感情中纠结着往前走，很多时候也会辗转打听着X的消息，偶尔听说有了男朋友，过得幸福！小小的失落…

在大学，Y尝试过去喜欢别人，总是觉得不够好，甚至都忘记自己要的是什么样的人，只是觉得都不够心目中那么美好…Y以为自己是深爱着那个安静的女子的，直到有一天听到了Z有了男朋友的消息…Y以为自己会痛不欲生，最后发现自己居然并没有想象中的那么痛苦。

大学毕业，大家各奔东西，开始了工作…Y和X的联系也很少，甚至是可以说很少很少…偶然的一次，和某友B的闲聊中听到了很多令人震惊的事，原来X的感情也一直很纠结，过得并不幸福，彼时的心居然像是吃过巧克力后的嘴里的的味道，微微的发苦，却是原来Y可以看着X和别人在一起，但是不能听见X过得不好…也许是这样隐晦的多次打探，换来了B的追问，终于那个没有被人洞悉的秘密开始让人发现了，慢慢地这个秘密被提及了，或者说是这一回被震惊的人是B，所以有了如下的对话…

B：好吧，我不得不承认你还是很厉害的，就这样？

Y:恩

B：首先我对于您的保密性表示绝对的佩服；其次我对于您的忍耐力和坚持力表示绝对的佩服；再次我对于您自己稀里糊涂追逐一个替身的后知后觉到极致的迟钝领悟力表示无语；最后尤为重要的是我对您时至今日都没有告诉她表示无语。

Y:我只是觉得，很遥远…

B：oh！苍天啊大地啊！我只见过给别人判死刑，没见过给自己判死刑判的这么彻底的。

Y:而且，不知道怎么开口？

B：您几岁了啊？怎么这么幼稚啊？

Y:我以为，这样的事情，是不能因为单方面的原因就判自己死刑的，因为人啊就只有一辈子，什么来生下辈子都是骗人的，如果错过了都会遗憾很久，更何况是这样不经努力就放弃的感情，如果真的不值得，那么你为什么直到今日仍然能够记得那么清楚，我见过很多求之不得的然后明智放弃的人，我也见过很多一直一直坚持固执地追逐一个人最后赢得胜利的人，无论哪一种人我都欣赏，至少那是自己努力过的，唯独你这样的人，什么都没做就搁置在这里，搁置就算了，有本事你就别再在意啊，真没出息！

Y:呃，我不敢…

B：从来我就知道，有什么东西如果你想要，那就得靠自己去争取，说得不好听，早死早超生总比半死不活来得强不是么？

Y:后果很严重的，或许连普通朋友都不是

B：但是你不试一下怎么就知道不成功了？

Y:那？我试一下？

B:为什么不可以？而且，我觉得你是真心的，我也希望X可以幸福！

Y：那要怎么做啊 ？我们都很久没有联系了，突然联系不会很怪异么？她会觉得我别有居心的.+.+.+

B：拜托，你难道不是别有居心么?而且，我们要的不就是她知道么？

Y：那我要怎么做啊？

B：我以为X是一个需要真心的人，所以你所有要做的就是真心的关心，仅此而已。。。

Y：具体一点儿？

B：这个是要你自己去琢磨的，ok？

Y：那以后有什么地方希望你多提点我一下。。。

。。。