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q******s
发帖数: 7469 | 2 How do you define 牛鼻
Met has about as good coverage of the world as British and Louvre, however
there are not many masterpieces.
British has Elgin Marble, Rossetta stone. The asian collection is primarily
in Victoria and Elbert museum.
Louvre definitely has the biggest collection and most masterpieces among the
three. However, some other museums has niche advantage over Louve.
For example, Uffizi has the biggest collection of Renaissant art, Prado has
an extensive collection of Spanish painters, |
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a*******r
发帖数: 3452 | 5 按圣经定义，上帝能力无穷大，人类即使能力从1到10000，比值还是零；另按圣经定义
，上帝在时间空间之外的，人类在时空内的杂耍，类似俺家母鸡在其笼里下蛋吧。
话说回来，还是很牛，下个指令得十几分钟才收得到吧。 |
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x****k
发帖数: 661 | 7 2009年就开始玩单反了，500D到2011年换7d，水平一直很臭，根本没入门，偶尔逛逛版
上看大师作品，总结的自己这辈子摄影没希望了。2014年底a6000 deal是3年来的唯一
摄影shopping。研究一下发现挺不错的，激发了新一轮的学习欲望。Xmas的时候就一直
看B&H video慢慢有一点idea，2015年几乎每个月都跑出去玩~
2月Everglades NP
都是7d拍的，everglades的wildlife确实不少~
5月Shenandoah NP
这张可惜的是日落的颜色我总觉得后期还欠一点，没能真实还原现场。
6月Arcadia NP
正是这张照片激发了我astrophotography的兴趣，之后一发不可收拾，每次去国家公园
都想在外面拍到很晚~每次总想万里无云的大晴天~
这是一个大家都照到滥到不行的地方（我后期下手可能有点重）：
8月Yosemite, Sequoia, Joshua tree NPs
这是sequoia起森林大火了，天空很厚的烟尘~
10月great smoky mountain
在山顶上开始deepsky imaging，第一次非常保守，... 阅读全帖 |
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x****k
发帖数: 661 | 8 赞！还有什么好target lz来写个总结吧。尤其是300-400能拍的。网上常讨论的M31,
M45, M33, M42, horsehead, rossetta, M8&M20, north american nebula, M81, Leo
triplet。
这次倒是去拍拍heart+soul nebula。 |
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j****c
发帖数: 19908 | 9 都被你写完了。。。
：赞！还有什么好target lz来写个总结吧。尤其是300-400能拍的。网上常讨论的M31,
：M45, M33, M42, horsehead, rossetta, M8&M20, north american nebula, M81,
Leo triplet。 |
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wh
发帖数: 141625 | 10 嗯，我后来想起来了，你说过去看rossetta stone的。那你去过tutenkhamen的墓吗？
我们中学有篇英语课文讲怎么偶然发现他的墓，说参与挖墓的人一个个都死了，说是法
老的诅咒。这一课我们学得很兴奋。大家都喜欢神神道道的东西。不过那个最先发现墓
穴的考古学家没死。 |
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I***d
发帖数: 1023 | 11 rossetta stone在大英博物馆吧。你不是在英国待过，应该看过啊。 |
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wh
发帖数: 141625 | 12 对我看过。上次小帕吹嘘他的语言学专业，说起rossetta stone，doha说她在埃及见过
复制的是不是？所以我昨儿发完帖后想起来我应该知道她去过埃及的。
你果然是对古董感兴趣。你不会懂希腊文拉丁文吧？我们这里有个叫gesund的人懂…… |
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a******e
发帖数: 6689 | 13 不仅讲得一般，每次去法国前都用Rossetta stone法恶补。
好看不好看这个看谁看了。书是全Zelda视角的。 |
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s********n
发帖数: 2939 | 14 我觉得如果你的蛋白能够用Ni-NTA column纯化说明N端的His-tag没问题，很大可能是
翻译提前终止或者降解了。如果是翻译提前终止的话，可能是稀有密码子的缘故，你可
以试试一些带有稀有tRNA的菌株，如Rossetta系列，也可以直接合成codon optimized
的基因。如果是降解的话需要分析一下是in vivo还是in vitro，in vivo很麻烦，可能
要换菌株，in vitro的话可以在做cell lysis的时候加protease inhibitors. |
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w**t
发帖数: 52 | 15 我遇到过好几次用Ecoli表达真核蛋白都是truncation，
应该是codon bias的问题，用Rossetta能好一些。
或者换insect cell。 |
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h**********8
发帖数: 650 | 16 你的问题我遇到过，几乎一模一样。
用的rossetta, 换了个培养基2yt，温度降到20度， iptg 降到0.2, ok 了。 |
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n***w
发帖数: 2405 | 17 Hi, all,
I am using Rosetta-Gami DE3 pLySs to expression my fusion protein which is
predicted as 91kDa.
I first did the expression assay and found protein expression was induced
after adding IPTG by testing the whole cell lysate. (bacteria at certain
time points, add 2X sample buffer, boil, SDS-PAGE).
Then I moved to culture more and lysed the cells using a sonicator, after
resuspending the pellet with 1x cold PBS/1% PI. After all these, I loaded
the supernatant and pellet side by side and stain... 阅读全帖 |
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T**********t
发帖数: 1604 | 18 pLysS只表达少量的Lysozyme用于抑制T7 RNA polymerase，而且表达出来的lysozyme在
胞内而不是细胞外，无法作用在细胞壁上。所以在lysis buffer里加lysozyme是必需的。
只不过我觉得lysozyme对于inclusion body本身的溶解应该没什么作用吧，它的作用估
计只是让细胞降解的更彻底，释放出更多的inclusion body而已。
of |
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s********n
发帖数: 2939 | 19 Then I moved to culture more and lysed the cells using a sonicator, after
resuspending the pellet with 1x cold PBS/1% PI. After all these, I loaded
the supernatant and pellet side by side and stained by GelCode and a bulk of
protein (I assume it was protein) was detected in the pellet part.
你有没有在supernatant中检测到你的target protein？如何检测的？WB or activity?
从你的表述你好像没有做WB。
Then I tried 1L culture and repeated the 2nd experiment. This time, I
incubated the supernatant with glutathione sepharose 4b beads an... 阅读全帖 |
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n***w
发帖数: 2405 | 20 Thanks. I did WB as well. Supernatants did show the specific band.
I will try freeze and thaw method rather than sonication tomorrow.
of
activity? |
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e****s
发帖数: 1125 | 21 那你加Beads以后的检测结果呢？WB检测的吗？ |
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N2
发帖数: 81 | 22 ask a question not relevant to this, R U from NANTONG?
of |
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n***w
发帖数: 2405 | 23 Yes. How do you know?
LOL |
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g*********r
发帖数: 9366 | 24 这一步要WB和commasie同时跑
大多数inclusion body的蛋白在WB 上也会显现，这个没什么意义
要看在supernatant 中有多少
而且要充分离心，取真正的清液
办法， 降温，少加IPTG，等等
实在不行denature |
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i***0
发帖数: 160 | 25 Microfluidizer or French Press will break the cell more efficiently compared
to Sonication. |
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n***w
发帖数: 2405 | 26 Thanks.
We don't have such device here =(
We are not protein engineering lab so my boss has no plan to purchase one in
the near future.
I repeated my experiments and modified my protocol a little bit and am
running gels now. Let's see how it
goes.
Thank you again!
compared |
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g*********r
发帖数: 9366 | 27 no, they can not "break" inclusion body
compared |
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N2
发帖数: 81 | 29 Check your mailbox at mitbbs!
of |
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i***0
发帖数: 160 | 30 For inclusion bodies, you have to reduce temperature and IPTG amount. One of
my proteins has inclusion bodies and very small amount of soluble protein.
But when I tried with 10 uM IPTG induction, the soluble part has about equal
amount of my protein compared to inclusion bodies. But when you have
efficient cell breaking method you will release almost all of the soluble
protein from the cells. So try with more expression conditions before you
enlarge culture size. Personally, I like French Press,... 阅读全帖 |
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g*********r
发帖数: 281 | 31 Maybe it is not necessary to ask, where did you put your GST? Normally N-
terminal GST will help your protein folding and solubility.
In term of inclusion body, like people mentioned, lower temperature and IPTG
concentration might help.
Or try some other Tag, like MBP. |
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w*********e
发帖数: 98 | 32 Try low temperature at 18 C, which should start right after you inoculate
the overnight culture into the final culture. Then wait until OD is 0.6-1.0,
add low concentration of IPTG such as 0.1 mM IPTG for 16-20 hours at 300rpm
. I just tried this protocol, it increased a lot the ratio of GST fused
protein in soluble fraction compared with that at 37 C. |
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n***w
发帖数: 2405 | 33 Thank you guys.
I use PGEX2T vector so GST is on the N-terminus.
I normally do 100ml culture size. Here is how I did this:
1. small culture of the bacteria from glycerol stock O/N, about 6-8ml.
2. transfer the small culture to 100ml 2xYTA medium in the next day morning
, shaking at 300rpm, 37degrees.
3. it ususally takes about 1hr 40min - 2 hrs to have an OD600 around 0.75.
4. add IPTG (stock 100mM) 100ul to get a final concentration of 0.1mM.
5. Lower the temp to 22degrees and shake at 300 rmp... 阅读全帖 |
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n***w
发帖数: 2405 | 34 Hi, how much did you inoculate into the final culture? 0.1mM IPTG is the
final concentration or the stock concentration? Thanks!
0,
300rpm |
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w*********e
发帖数: 98 | 35 I inoculated 5ml overnight culture (37 C)into 30ml medium because I was
using 18 C to shake ( cell grows much slower than 37C) until OD is 0.8 and
then I added IPTG at final 0.1mM. The speed of shaker when I started using
18 C is 350 rpm.
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w*********e
发帖数: 98 | 36 I used 30ml because I was trying to see if my protein could be expressed.
After IPTG was added, the culture was shaked at 18 C for 20 hours in order
to get enough cells.
The big difference based on your protocol is that I started low temperature
the next morning when I did final inoculation, while you still used 37 C.
I compared the soluble and the pellet fractions at 18, 28 and 37 C,so I know
18 C is the best one. |
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A****w
发帖数: 244 | 37 There are a few strains of E.coli(DE3). Which one are you using? I found
Rossetta 2 cells (DE3) is better in my case. As mentioned, low temperature
and low iptg will help expression. Auto-induction is another way to try. Or
leaky expression??
It's hard to say things wrong in expression, even if A and B share >90%
similarity.
Good luck. |
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h**********r
发帖数: 671 | 38 这个真不知道。不过BL21(DE)3的rossetta系列都有额外的tRNA,在一个p15 ori的低拷
贝质粒里，名字一般为pRARE或者pRARE2. |
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C*******e
发帖数: 4348 | 39 谢谢
我其实上周刚刚试了Takara家的一套5个chaperon plasmids
很可惜
因为用chaperon plasmid不能用codon plus或者rossetta strain
A蛋白完全没有表达
（WB探测不到；以前已经知道A有好几对tandem、甚至triple rare codon，需要codon
plus才能overexpression）
所以现在需要codon optimized ORF才行
还会继续试的
我想问问，如果说in vitro, low concentration试验的话
用什么手段检测/定性复合物呢
, |
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