f*********p
发帖数: 13 | 1 I do not think you fully understood my answer to your question yet. let me put
it this way.
what kind of BL21 are you using? BL21 has BL21 (DE3), (DE3) pLysS, ...
different subtypes. I assume that you are using DE3 related strains. DE3
strain has a lambda phage lysogenized in BL21 e.coli, on which there is a T7
polymerase gene under the control of lac promoter. when you grow the cells up,
no or very little T7 pol can be expressed. when you add IPTG, it induces the
expression of T7 pol, which the |
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c******a
发帖数: 267 | 2 Nature Genetics Vol32, Oct,2002 p306P311
Human securin interacts with p53 and modulates p53-mediated transcriptional
activity and apoptosis.
The first report show the relevance between human securin and p53.
The gene PTTG1 is considered human securin gene which can inhibit
sister-chromatid separation in vertebrates, thus may contribute to cell-cycle
and tumorigenesis. The authors used phage display to identify the interaction
between hPTTG1 and p53, and they carried out a series of functional a |
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l*****k
发帖数: 587 | 3 This is a pretty cool paper,
1. The experiment they used to show securing binding of P53 affects ability of
P53 to bind DNA, I think it is a long shot to have that conclusion, there are
plenty of other alternative explanations. However, their in vivo experiments
are very convincing.
2. Can anyone tell me how many AA can phage display vectors successfully
express?
A recent paper by ES. Lander and TR. Golub in nature genetics (Jan 2003) “A
molecular signature of metastasis in primary solid tumors” |
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d********r
发帖数: 303 | 4 HI, all,
I eventually find a second paper for our random journal club. I first
randomly pick a letter from S, N, C and P, representing Science, Nature, Cell
and PNAS. I got C. Then I choose a month. It is 04/2003. There are two issues
in that month. I use a coin to pick one. It is the 04/18 issue. I go to its
preview. There are four of them. I randomly choose one. It is the second one,
named as: Global Phage Diversity (Cell, Vol 113, 141, 18 April 2003).
Here is the citation and summary of that |
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c*******e
发帖数: 5818 | 5 Here I assusme what you ask is simply original reversal mutation, otherwise it
will be complex 'cause the reversal one could be second mutation on the same
gene or totally different gene (mostly tRNA genes). So, We can roughly
estimate it like this. If spontaneous mutation rate is 10 to -6, the chance of
reverse rate could be 10 to -6*2, so it will be 10 to -12. See, very very low.
I guess all the calculation about mutation rate is from bacteria or phage. It
is impossible to use high orgnism, so |
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p*****m
发帖数: 7030 | 6 看你怎么理解吧 我倒是觉得国内这些年进步很明显。你想一步到位这个也不现实啊 只
要它在变好就行。我是觉得不管是聘用制度 经费制度还是回去的人的质量 这几年看着
都还是很有希望的。当然了 长远的事情谁也没办法预测
说到这个人治的问题 我也觉得一流学术机构的成长牛人的作用肯定是第一位的 有了牛
才会有相应的制度 没有牛有纸面的制度也是白搭。MRC没有Bragg和Peruz只怕就是两个
样子 CSHL当年要是没有phage那一票人可能也不会是个一流的研究机构 |
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s********n
发帖数: 2939 | 7 Mass spec, phage display, FPLC, HPCL, high throughput XXX... |
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v**********m
发帖数: 5516 | 8 看看这个版上的生物问题水平，这些人能去工业界吗？真能去工业界的不会在这些问题上打叉的。
我买卖过医学生物公司的股票，所以对这个行业有些了解。
我奉劝各位学生物的，多去掌握一些比较靠脑子的生物技术，如蛋白人工修饰，纯化，
计算机辅助的药物设计，phage-based 大分子相互作用筛选优化技术，分子相互作用模
拟，三维结构解析，临床实验数据统计等在制药公司研发中正真实用的技术，不要去片
面追求能发Fancy文章的课题，那些东西对你将来进工业界是没有用处的。
通常而言，越能发好文章的领域离应用就越远，甘蔗没有两头甜。
我随机找一个公司招聘的广告，看看吧。 |
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v**********m
发帖数: 5516 | 9 看看这个版上的生物问题水平，这些人能去工业界吗？真能去工业界的不会在这些问题
上打叉的。
我买卖过医学生物公司的股票，所以对这个行业有些了解。
我奉劝各位学生物的，多去掌握一些比较靠脑子的生物技术，如蛋白人工修饰，纯化，
计算机辅助的药物设计，phage-based 大分子相互作用筛选优化技术，分子相互作用模
拟，三维结构解析，临床实验数据统计等在制药公司研发中正真实用的技术，不要去片
面追求能发Fancy文章的课题，那些东西对你将来进工业界是没有用处的。
通常而言，越能发好文章的领域离应用就越远，甘蔗没有两头甜。
我随机找一个公司招聘的广告，看看吧。 |
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l**********n
发帖数: 201 | 10 当然。偶 lab 刚来了一个 tenured professor, taking his sabbatical，纯粹为了多
学点
新东西，就跟当年 seymour benzer 去 caltech 学 phage 一样。
但问题是现在学校研究所，中美一样，不会再有这么“奢侈”的机会让给年轻 PI 们了。
点好了才放心来吧。不过那个时候说不定你又不想来了。 |
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w******e
发帖数: 1187 | 11 你说用cpp deliver protein 做ips那个？我其实一直不太理解这个套路的
publication。cpp delivery历史很久了吧？要deliver的protein也是知道的。
就像去年nature chemical bio那篇in vivo SELEX一样。人家in vivo phage
display都做了n年了，换到SELEX上，那么poor的performance，也发baby
nature，想不通啊～ |
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w******e
发帖数: 1187 | 12 看来大家对in vivo SELEX这个phrase的理解不一样呵呵。我是套用in vivo
phage display的意思 |
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W****C
发帖数: 1937 | 13 phage display 做这么多年了 筛到几个有用的东西？ 还有在裸鼠体内筛的， ｐｈ
ａｇｅ这么大的东西在体内筛有意义嘛？ |
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W****C
发帖数: 1937 | 14 phage display 做这么多年了 筛到几个有用的东西？ 还有在裸鼠体内筛的， ｐｈ
ａｇｅ这么大的东西在体内筛有意义嘛？ |
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h*****1
发帖数: 26 | 15 大家好我是一个外行，
Lambda phage DNA: Escherichia coli W3110
这里W3110是什么意思？
谢谢！ |
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m*****a
发帖数: 26 | 16 search in ABRC, possibly you can not get such a very good library since most
are just all-in-one in phage. |
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w******e
发帖数: 1187 | 17 嗯我就知道哪些热，但不知道是过去现在还是将来时。。。比如nanomedicine吧，
也炒了10几年了，今年去AACR，发现没几个poster做nano的。。。targeted
delivery也是。。。搞得我严重怀疑自己是不是太被big picture吸引，跳进了
一个纯忽悠的领域。。。
also，bme到底是个什么概念？microfluidics算吗？library screening methods
(phage display, SELEX, etc)算吗？biosensor算吗？我很困惑呵呵 |
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w******e
发帖数: 1187 | 18 对ligand-receptor interaction的polyvalency一直很困惑：理论上targeting
ligand要通过polyvalency来提高apparent kd，就要ligand和receptor的
positioning和density恰好match，像齿轮一样嵌合，才能有效。可是ms
在NP上做polyvalency的人很少会去根据receptor的具体情况做一堆计算然后
决定如何incorporate ligand，polyvalency带来的好处也是从几倍到几万倍
不等。我的问题是，到底有没有组做rational polyvalency design？理想
状态下（比如3个ligand跟3个receptor同时完美结合），polyvalency对
apparent kd的贡献有没有个公式？最后如果可以很容易的通过polyvalency
提高kd和specificity，为什么还要强调antibody/aptamer的kd？搞几十个
phage display选出的peptide行不行？
请牛人指点（给个link什么的也成）。//bow |
|
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a********e
发帖数: 669 | 20 概念上的，感觉就是venter的institute比较牛
其他人不就是在mutate E.coli或者phage的metabolism搞石油就是在搞peptide设计
理论上没有太大的突破 |
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a*****a
发帖数: 312 | 21 自己手动选了一个primer，lambda phage DNA作为template，
PCR结果有两条带，一条长度是我要的，一条的长度为一半。
有什么软件可以用来搜索template，看有没有跟我的primer类似但不一样的序列呢？ |
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a*****a
发帖数: 312 | 22 有一段oligonucleotide，我需要知道在lambda phage里面有多少相似(可以不完全一样)
的序列。
有什么free软件可以用吗？ |
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n****r
发帖数: 37 | 23 急用
多谢拉
Simultaneous expression of glutaryl-7-aminocephalosporanic acid acylase gene
and lysis genes of phage λ in a recombinant E. coli
麻烦发到m*****[email protected] |
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b******r
发帖数: 111 | 24 I am cloning 3.4kb human genome DNA into a 8.4kb vector.Isolate some
plasmids. Recut,then 3.4kb bands
appear. But sequencing shows it is not 3.4kb human DNA,it is a unknown
fragment. Strangely,the size is same
as 3.4kb! What happen? Any idea to eliminate recombination ? The competent
cells used are Invitrogen ElectroMAX™ DH10B™ T1 Phage-
Resistant Competent Cells Cat. No. 12033-015. |
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p*****m
发帖数: 7030 | 25 这个是两码事。一个牛人做一个领域做到功成名就换个方向是常事，这不说明伊换的就
一定是对的呀。事实上fish field近30年没有贡献特别巨大的breakthrough，这个话可
不是我说的；相比之下，phage, ecoli, yeast,worm，fly,sea urchin,arabidopsis,
mouse,blabla 甚至monkey，这些个model system都贡献过超出系统本身的影响啊。 |
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H****s
发帖数: 301 | 26 I am not really sure what you mean by your question. If you want an antibody
that only binds to your target protein, but not the homologous protein, you
can design your phage/yeast selection and downstream screening strategies (
you get what you select for).
If you already have an antibody that recognizes both your target and the
homologous negative target, you can engineer the antibody to keep
specificity. A good screening assay/strategy is a must here.
If I can be of any further help, please l... 阅读全帖 |
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H****s
发帖数: 301 | 27 很抱歉。这两天比较忙，关于抗体工程这个领域，一时半会说不清楚，因为不仅仅是学
术研究问题。如果你对这个领域有点兴趣，建议你pubmed一下以下几个人写的综述：
Richard Lerner，John McCaffery，Geoege Smith， Gregory Winter， Andrew
Bradbury和James Marks。这几个人基本上是antibody phage/yeast display领域的开
创者，并且也是抗体工程以后发展的方向。如果你对某一个人特别感兴趣，请告诉我，
我再详细给你说关于这个人的一些情况（学术研究方面的）。 |
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H****s
发帖数: 301 | 28 一个比较简单的法子，把这段蛋白的基因随机片段化（最好是50-100bp大小）。然后把
片段文库克隆到噬菌体展示载体（phage display vector）上去，用你的抗体来筛。把
筛到的克隆测序，除去假阳性，然后做序列比较，（一般来说）你就可以找到抗原表位
了。 |
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F*******2
发帖数: 103 | 29 想问一下，直接用genomic DNA (lamda phage 的genomic DNA)做PCR模版，pcr难度大
吗？会做不出来吗？ |
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a*****y
发帖数: 269 | 30 sent to my mailbox. information not confirmed
Biologist Protein Engineering
San Diego, CA
$70K - $100K
10% Targeted Bonus
Relocation Assistance
Masters Degree (NO PhD's)
The right candidate must have strong skills in molecular biology techniques
and/or biochemical assay
development.
Biologist
San Diego, CA
$70K - $100K
10% Targeted Bonus
Relocation Assistance
Masters Degree (NO PhD's)
Experience in antibody discovery and engineering, phage display technology
or protein structure and
biochemistry... 阅读全帖 |
|
A******y
发帖数: 2041 | 31 Are those avoid context dependence of DNA sequence specificity? I mean zinc
finger protease generated from phage display can recognize 18 base pairs
which is the minimum base pairs you need... |
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t****x
发帖数: 1429 | 32 很有可能是 phage contamination. 因为OD会下降，出现白色絮状物。 不过不知道为什么过夜还
能长到1.5， 而且加了IPTG， 有时候还能拿到蛋白。 |
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C*********m
发帖数: 213 | 33 春天来了，虫子多了。你的应该是Phage 污染了。
M9培养基中，37度摇到OD1.0后加IPTG诱导。
6的时候就突然开始猛降，培养基也变得澄清，OD值降到大概0.1。如果接着摇OD值还能
再升回来，摇过夜的话还能升到1.5左右。培养基中有很多白色絮状物，闻上去不像大
肠杆菌的味。这个时候加IPTG诱导，有时能拿到自己的蛋白，有时候拿不到。 |
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w******e
发帖数: 1187 | 34 A system for the continuous directed evolution of
biomolecules
Kevin M. Esvelt1, Jacob C. Carlson2 & David R. Liu2,3
Laboratory evolution has generated many biomolecules with
desired properties, but a single round of mutation, gene expression,
screening or selection, and replication typically requires days or
longer with frequent human intervention1. Because evolutionary
success is dependent on the total number of rounds performed2, a
means of performing laboratory evolution continuously and
rap... 阅读全帖 |
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w******e
发帖数: 1187 | 35 mRNA/ribosome/phage/bateria/yeast/microbeads/micro droplet display的铜锈们
都去什么会啊？
只知道有抗体相关的会，那做peptide/alternative scaffold/enzyme evolution
的有啥类似的会否？gordon系列有这个topic否？
多谢！ |
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s******9
发帖数: 283 | 36 问几个问题。好奇yeast display能稳定表达多大的protein？Phage display好像只能
表达不大的peptide。Microfludics做IVC有多成熟？是不是都只有fluorescence
readouts？ |
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s******9
发帖数: 283 | 37 还有，象ribosome/mRNA display这些single-molecule based display和yeast/phage
display在传统的screening环节相比是劣势还是优势？ |
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l******a
发帖数: 3339 | 38 你是说yeast surface display？貌似以前读过一篇review，比起phage之类有很大的
technical challenge，不是人人都能做的。 |
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l*********1
发帖数: 351 | 39 要做大蛋白就用mrna或者ribosome display.不需要优化phage或者yeast 表面的蛋白表
达.
缺点就是贵,很贵,二screening稍微麻烦点,时间稍长. |
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s******9
发帖数: 283 | 40 好像抗体还是phage display用得多， 所以我好奇ribsome display在screening相比上
肯定会有弱点。 |
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w******e
发帖数: 1187 | 41
not true on PIII. scFv is the tour de force for phage hehe
there are many fancy types, but fluorescence is so nice why change?
btw many other displays also do fluorescence. |
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w******e
发帖数: 1187 | 42
phage
very tech sensitive |
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w******e
发帖数: 1187 | 43 Thanks for the input.
en the variability on display level is indeed daunting.
BTW why didn't bacteria display take off? bacteria can do FACS too,
right? plus I assume it's easier to maintain.
Also, IP issues seem to really hurt protein engineering in general,
according to Bradbury's recent review. I assume phage display is
even worse? considering there are mutliple big shots hehe |
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w******e
发帖数: 1187 | 44 check this out: http://www.xconomy.com/boston/2010/11/30/adimab-finds-4m-
more-from-google-and-other-investors/
I know it has potential, but didn't know it's already cashing in...
is any phage display company doing so well, except ImClone?
Well the sad thing is, if sth is already the darling of VC, it may not
be a good target for technology innovation any more... go work on sth
fancier and pray to god it'll be the next big thing? or work your way
into those rising star companies and sit tight fo... 阅读全帖 |
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H**********J
发帖数: 351 | 45 Assay,Group Leader;Roche; Shanghai
1) A Ph.D. in biochemistry, molecular biology, cell biology, biophysics
or related field
2) 5-10 years pharmaceutic/biotech research experience.
3) Proficient in biochemsitry, enzymology, molecular biology, cell
biology, protein production assay development and HTS
4) Demonstrated proven experience, of an innate sense of urgency, in
managing projects, ability to deal with ambiguity, appreciation of
priorities and critical factors
5) Excellent le... 阅读全帖 |
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h******i
发帖数: 1 | 46 For more information about this exciting opportunity, contact Fei Xu (xufeih
@gmail.com) or Professor Raymond Stevens (s*****[email protected]).
DIRECTOR, ANTIBODY DISCOVERY
RuiYi is a newly established biotech company in Shanghai, China, focusing on
discovery and development of novel antibody therapeutics against GPCR
targets. The company’s core technology overcomes previous hurdles of
antibody generation against GPCRs, unlocking this important target class for
the development of highly specific an... 阅读全帖 |
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n***3
发帖数: 663 | 49 Curr Protoc Immunol. 2009 Aug;Chapter 9:Unit 9.8.
Phage display selection, analysis, and prediction of B cell epitopes.
Freund NT, Enshell-Seijffers D, Gershoni JM.
Please send the paper to e*********[email protected]
Thanks! |
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m*********e
发帖数: 378 | 50 我真的只会一点点的并没有写上来啊，银染，southern, northern, caspase activity
, pgp activity, phage display, yeast knock out, point mutation, 果蝇酵母的都
没写的
其实是现在掌握的很好：从前掌握的很好（现在不做但是也能很快捡起来）：现在掌握
的一般1：1：1吧。
我本科的training很辛苦很全面，在实验室整整作了两年的intense banch work，提到
的技术大部分都在那两年学的，当然掌握程度不一样是有的。到了这边自己做课题，有
的技术磨的更好，有的也就荒废了
在这边死在了不大考谱的课题上，就转硕了，training也是照着PhD上的。所以不是一
定要比所有的PhD会的技术少呀。
western, protein expression in mammalian cells, flow cytometry of TUNEL
assay, enzyme assay, GFP, cell and small animal handling 是现在经常做的
realtime, PCR... 阅读全帖 |
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