n********k
发帖数: 2818 | 1 meaning whether the in vitro insight you gather will hold true in vivo...and
how robust the in vitro system could be...if it is very robust and highly
representative, and have the potentials to offer novel insights, then it is
very much valuable, otherwise it is hard to say...Taking cancer field as an
example, tons of the in vitro insights from earlier days turn out to be
misleading or in vitro artifacts... |
|
n********k
发帖数: 2818 | 2 meaning whether the in vitro insight you gather will hold true in vivo...and
how robust the in vitro system could be...if it is very robust and highly
representative, and have the potentials to offer novel insights, then it is
very much valuable, otherwise it is hard to say...Taking cancer field as an
example, tons of the in vitro insights from earlier days turn out to be
misleading or in vitro artifacts... |
|
c******r
发帖数: 3778 | 3 选择in vivo还是in vitro system除了大家都知道的优缺点之外要考虑的还有一个问题
，就是目的是什么。
如果是一个成熟的project，已经有很多positive的pilot experiments，有很多in
vitro的data，需要证明in vivo的relevance，那么确实选用动物实验会更好，更有意
义。
但是如果是一个摸索性的前期实验，根据成败来决定project的概念是否成立。那么可
能选用一个更简单，快速的办法更有效。这时候如果直接上老鼠，不但价格贵，程序复
杂，手续繁琐，时间太长。如果失败risk就比较大。而即使成功，由于没有很多in
vitro的test没法同时进行，浪费很多时间。
lz这个，我看还处于project的早期阶段，还不知道是否能够做下去。用个简单的in
vitro实验摸索一下，可能比较合适。等比如说系统建立了，证明某个因子是介导这一
过程的，然后去in vivo系统里面confirm，可能更好一些。
and
is
an |
|
J****S
发帖数: 15 | 4 Pls contact : d******[email protected] for more information
Report to: Head of in vitro Biology
Description:
Looking for a highly experienced and motivated Scientist with a strong
cellular and molecular pharmacology background to lead the In Vitro
Pharmacology group.
The successful candidate will play a leadership role in the company
including building up an internal team, leading projects in a
multidisciplinary team, as well as efficiently interacting with clients to
drive project success.
The can... 阅读全帖 |
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s******y
发帖数: 28562 | 5 Promega, TnT T7 coupled transcription/translation kit
Depending on what degradation system was responsible. In vitro translation
system also contain ubiquitin and proteasome. However, it does not have
lysosome or golgi (which also contains protein degradation machinery).
You can add 10uM MG132 in the in vitro translation system (or your 293T cell
culture medium) to inhibit the proteasome.
的系统更容易降解我的蛋白？
Yes. If you want, you can try bacterial in vitro expression system. BUT, it
won't contain the |
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p*****n
发帖数: 981 | 6 thermo scientific刚刚推出了human in vitro protein synthesis system
我大概看了一下，貌似很不错
不过我没用过。
只不过几乎所有in vitro系统表达量都不高（或者说要高表达就要花好多钱） |
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h**********y
发帖数: 18 | 7 我做过一个实验，不是MAPK，用的是MAP2K，
发现在in vitro kinase的条件下，MAP2K自己就可以autophosphorylation，我猜想
MAPK应该也会是类似的情况，毕竟体外的实验总是有一些artifact。另外，看早期的
MAPK的paper，他们的in vitro kinase assay是不需要加入MAP2K的。当然加入MAP2K之
后，对MAPK底物的磷酸化会强很多。
另外，你在你的kinase里面有没有加入DTT？我们发现某些kinase因为某些特定位置的
aa，对DTT是敏感的。而ERK在相应的位置恰好存在一个这样的aa |
|
A******y
发帖数: 2041 | 8 Where do you get your HDACs? Also, which HDACs? The in vitro data for most
class IIa HDAC are wrong...including HDAC8. Class IIa and HDAC8 do not
deacetylate the acetylated substrate (or very little)...they do bind though.
I can give you an activity list...
Class I (Ac substrate)
HDAC3 > HDAC2 > HDAC1
Class IIa (TFA substrate)
HDAC8 > HDAC4 = HDAC5 = HDAC7 = HDAC9...
Class IIb (Ac substrate)
HDAC6 not so active but better than HDAC1
HDAC3 is at least x100 time more active than HDAC1/2 in vitr... 阅读全帖 |
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k****n
发帖数: 158 | 9 小可想，定有很多实验室或者phd把大部分精力投入到in vitro实验中，但是目前如果
没有in vivo的实验似乎很难发表很好的文章，所以请教怎样能做好in vitro的实验，
从立意的高度到具体的实验方法，望各位大侠分享下经验。 |
|
r*****t
发帖数: 4793 | 10 我试了用400 ul extraction buffer 处理差不多150ug cell pellet, 然后用90ul做
好的cell extract 和10ul in vitro transcription/translation的蛋白混合，37度
反应， 到4小时都没大变化
我现在不知道哪个因素是比较重要的？我这个条件是cell extract 不够么？ cell
extract 里面要加些SDS NP40什么的吗？（有的protocol不加）。还有的说in vitro
transcription/translation的蛋白要新鲜，这个很重要么？ |
|
y******n
发帖数: 8667 | 11 用TIRF or fluorescence microscopy来研究tubulin dynamic in vitro regulated by
other tip proteins.
But now I met a lot of problems:
1. Is labeled tubulin and tubulin easy to lost their activity in vitro?
2. What conditions is better for their polymerization?
以前看到过很好的GMPCPP seeds， 但之后再也重复不出来了。同一批labeled的
tubulin蛋白好象都形成一些很亮的点，应该是蛋白凝聚了或变性了。但新labeled 的
tubulin 也不行。有人有什么建议吗？ |
|
m*****d
发帖数: 566 | 12 你是说in vivo 和in vitro范围太大吗? 具体一点，就是in vitro的这个部门主要做
SAR profiling，很多的counter screen, biochemical, cellular assay 都有。in
vivo这个部门主要做animal sample，也是各样的assay, 具体是什么assay我还不知
道。 |
|
v**********m
发帖数: 5516 | 13 XOMA 052 Shows Potent In Vitro Inhibition of Interleukin-6 Production in
Human Myeloma Cells
BERKELEY, Calif., Apr 19, 2010 (GlobeNewswire via COMTEX) -- XOMA Ltd. /
quotes/comstock/15*!xoma/quotes/nls/xoma (XOMA 0.80, +0.08, +11.30%) , a
leader in the discovery and development of therapeutic antibodies, announced
that independent researchers today presented results showing that XOMA's
antibody to interleukin-1 beta (IL-1 beta), XOMA 052, was highly effective
in reducing production of a protein ... 阅读全帖 |
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m****g
发帖数: 530 | 14 Principal Scientist / Team Leader, In vitro pharmacology : Shanghai, China
Employer:
AstraZenca Pharmaceutical Innovation Center China
Website:
http://en.astrazeneca.com.cn
Location:
Shanghai, China
Posted:
August 11, 2009
Expires:
October 11, 2009
Jobs by tag(s):
astra zeneca astrazeneca principal scientist leader in vitro pharmacology
shanghai china discovery
biological Phd cancer
Requisition number:
SHA0000005X
Science jobs from AstraZenca Pharmaceutical Innovation Center China: job
descripti |
|
c*****x
发帖数: 405 | 15 【 以下文字转载自 Biology 讨论区 】
发信人: civiclx (whoknows), 信区: Biology
标 题: 问一个cardiomyocytes 和skeletal msucle cells in vitro culture的问题。
发信站: BBS 未名空间站 (Thu Apr 29 12:16:29 2010, 美东)
刚刚开始搞这个体外培养，有些不懂的地方。
这两种细胞要是没有外界刺激，比方说applied electronic field，平时会不会自己
contract?
是不是都要外界刺激才会contract呢？以前有个教授告诉我心肌细胞只有外界刺激才会
收缩，自己收缩是要快死的细胞。可最近看了一篇文章又说心肌自己会收缩，骨骼肌要
外界刺激才会。可我自己培养的时候看到骨骼肌没有刺激的时候也轻微颤动。所以糊涂
了。多谢。 |
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y********a
发帖数: 397 | 16 造人运动-IVF(in vitro fertilization, 体外受精) in the L world
你和你的爱人排除万难下定决心决定要一个维系你们感情的纽带,可爱的小baby. 下面
该如何走呢?
第一步: 联系一个fertility center, 选择很多,这个最好咨询你的妇科医生或家庭医
生推荐一个比较decent离家比较近的地方.
第二步: 医生会做一些基本的tests并考虑你和你爱人的年龄和身体状况来帮助你们决
定用谁的卵子(egg)与sperm(精子)结合制造受精卵(embryo), 用谁的子宫(uterus)怀孕
.
第三步:医生皮下注射卵巢滤泡刺激素( follicle stimulating hormone)给egg donor
, 刺激尽量多的eggs 从卵巢(ovary) 成熟. 最理想要收获至少8-15个eggs 才能确保较
高的体外受精成功率. (自然状态下女性每月只会有1-2个卵子成熟).这个过程大约要14
天, 每两三天就要到中心去抽血照超声波来监控成熟情况.
第四步:收集eggs. 这个procedure是在全麻下进行的, 在经阴道超声波( |
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y********a
发帖数: 397 | 17 Option #3: ML with your decent good looking male friend, done! no measure of
the temperature or the collect of the sample. LOL, just kidding.
You are right, Zakuro, 我的第一个post是只谈了一个 体外受精的option, 最艰难最
花费最technical的一个option. 因为有些女同希望用一方的卵子受精,用另一方的子宫
怀孕来建立一种connection. 这时in vitro就是唯一可行的办法了.
你说的这个option2, 也最好是在医院里由医生来操作吧.
我的post大家仅作参考了,要想得到全面详尽的介绍, 还是要咨询你的医生. |
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g*******u
发帖数: 107 | 18 有人用过 Nova In Vitro Fertilization: Fertility Clinic in Mountain View, CA
？？ 不知道这个医疗服务好不好？ |
|
q**********0
发帖数: 335 | 19 Recenly I use T3 megascript do in vitro transcription. The results is always
bad, the OD260/280 is about 1.53. A big white pellet can be seen after
isopropanol precipitation and RNA concentratin measure is about 1.0 ug/ul.
However, when run a RNA gel, the band is very faint even using 5 uL. I don't
know why? Maybe there is protein contamination? or RNAse digestion? Please
help. Thanks a lot! |
|
a****k
发帖数: 1130 | 20 We normally do overnight reaction for in vitro transcription. Make sure your
DNA template is good.
always
't
Please |
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i*****i
发帖数: 30 | 21 这里有几个概念：
1. in vitro expression: myc-tagged protein-A IP Flag-tagged protein-B 这是普
通IP过程。
2，如果分开表达的WHOLE CELL LYSIS做IP，那说明这2个蛋白相互作用更加直接(对上
下游信号要求不严格)，很可能是一级结构结合。
反之，就是需要正确折叠或者磷酸化修饰等二级三级结构。
3. 一起转染表达的话，根据表达量来决定IP需要的WCL ug数.最常见IP：400-500ug
WCL加1ug抗体。 |
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m*****d
发帖数: 566 | 22 照现在的制药业状况，大药厂的研发部门，是做in vivo前景好些，还是做in vitro前
景好些? 我知道discovery整体就不乐观，但是我只有discovery(biology, biochem,
HTS) 方面的background. 面临选择，请大牛给说说? |
|
c**a
发帖数: 94 | 23 就是Histone acetylation/deacetylation,蛋白放一起做in vitro assay. 有没有什么
trick啊？买来的recombinant protein都不管用，positive control都做不出来。不知
道到底
哪里出了问题。 |
|
n********k
发帖数: 2818 | 24 I think the harder part is not to establish a system but its relevance to in
vivo...in the related research areas but haven't followed this line much...
and for most of part, it is kind of a 20 century approach...that said,
people/I would love to see a robust in vitro system which could be used to
model in vivo in a meaningful way...BTW, there are tons of advantages coming
with such a system if a relevance could be established/validated...
化, |
|
w*******r
发帖数: 23 | 25 Exactly, that is what I am thinking about.
Besides, I think another advantage of this in vitro system is that you can
isolate and purify related factors more easily by biochemical methods than
in vivo. |
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f****n
发帖数: 114 | 26 小弟最近在做RNA in vitro transcription，用的是ambion 的MEGAscript kit。
可是不论是用PCR产物直接做模板，还是连接到质粒上再线性化做模板，都得不到sharp
的条带，size比预期小很多并且有拖尾，但kit里阳性对照可以出来。
已经排除了RNase污染。估计是DNA模板纯度的问题，请问各位PCR产物用啥方法纯化？
另外，我在T7promoter序列前面加了7个碱基，难道是这个影响酶的效率？
请各位高手帮忙！ |
|
h**********r
发帖数: 671 | 27 Title:
In vitro 'sexual' evolution through the PCR-based staggered extension
process (StEP)
Nature Protocols 1, 1865 - 1871 (2006)
Published online: 22 November 2006 | doi:10.1038/nprot.2006.309
Email: m******[email protected]
Thank you so much! |
|
s******s
发帖数: 13035 | 28 要做大量的Biotin label的RNA fragment （大概几百bp)，做类似
in situ的实验，大家都用什么kit啊？
我只用过roche的nick translation mix做过DNA的probe, 这个RNA
的是不是质粒前面有个T7/73/sp6就可以了？in vitro transcription
怎么终止啊？还是必须把DNA切下来转录到底？ |
|
d****i
发帖数: 2346 | 29 我们用的就是第一种方法，蛮好的，我没觉得有什么问题。因为就算是这个plasmid的
所有sequence都被methylated了也无所谓，转到细胞内以后只关心我的promoter和
luciferase的转录表达，其余backbone上的序列不要也无所谓，有没有转录表达没关系
。再说你肯定要做一个control。
第二种方法看上去确实是更严谨，但是我个人认为和luciferase转录表达有关的，来自
plasmid的只有promoter和后面的polyA了，不一定对啊。
如果你实在不放心可以去要这个质粒：(pCpGl-basic)。backbone上都是修饰过的没有
潜在methylation位点的。整个plasmid拿来做in vitro methylation对backbone没有影
响。
http://www.ag-rehli.de/materials.htm#pCpGL |
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X******n
发帖数: 24 | 30 想做 PKR in vitro kinase assay, 从没做过这类试验，请帮忙推荐一个好用的 kit.
非常感谢！ |
|
X******n
发帖数: 24 | 31 想做 PKR in vitro kinase assay, 从没做过这类试验，请帮忙推荐一个好用的 kit.
非常感谢！ |
|
g*****n
发帖数: 241 | 32 我觉得我做的一个E3 ubiquitin ligase可能可以ubiquitinate另一个蛋白，想做个In
Vitro Ubiquitin Assay验证一下。但我们实验室不做相关的实验，想找大家推荐一个
性价比比较高的kit。
谢谢 |
|
|
l**********1
发帖数: 5204 | 34 Are you sure you can use only fluorescence microscopy来研究tubulin dynamics
in vitro regulated by other tip protein?
http://www.mitbbs.com/article_t1/Biology/31629913_0_2.html
27th floor
If not,
please just go to
//cryoem.berkeley.edu/microtubules.html
//cryoem.berkeley.edu/cryoem
//cryoem.berkeley.edu/microscopes
more just click all icons you can open that web sites.
//mcb.berkeley.edu/index.php?option=com_mcbfaculty&name=nogalese
then rethink about which kind of instrument will be needed?
by
的 |
|
s****n
发帖数: 234 | 35 I think the in vitro tubulin dynamics experiments using TIRF microscopy will
be able to say whether the presence of tip protein(s) affects the tubulin
rescue and shrink rates. A complete understanding of the underlying
molecular mechanism may require structural studies using other methods
including EM.
dynamics |
|
h******y
发帖数: 1374 | 36 请问在in vitro里有啥好方法测量肿瘤细胞的fatty acid metabolism和fatty acid
oxidation?
谢谢！ |
|
u***e
发帖数: 71 | 37 anybody know how to measure insulin biological activity in vitro? |
|
y*****0
发帖数: 15 | 38 向各位大牛请教：为什么各种in vitro transcription的 kit 价钱差这么多？in
vitrogen 的 mMESSAGE mMACHINE® T7 Transcription Kit $428（25 reactions
），而 Fisher 的 TranscriptAid T7 High Yield Transcription Kit只要 $242(50
reactions). |
|
z*********s
发帖数: 38 | 39 我们最近在做一些In vitro Ubiquitination Assay。实验做完以后，需要把修饰底物
的泛素再移除。我们不知道哪一类去泛素化酶是对应于我们的底物的。请问在体外有没
有通用的去泛素化酶，可以移除任何底物的泛素化修饰。 |
|
D******K
发帖数: 557 | 40 USP2 catalytical domain shows the most robust non-specific DUB activity in
vitro. |
|
i*****e
发帖数: 633 | 41 生物外行。昨天和朋友聊天，说到老鼠癌细胞可以在碟子里养，还自己三维生长。可是
来自病人的癌细胞在碟子里活不了，只能注射到老鼠身上。癌细胞不都是疯长的和细菌
一样吗？很好奇，为什么人类干细胞啊 血脑屏障细胞 肝细胞之流都可以in vitro，而
人类癌细胞却那么弱？ |
|
r**********e
发帖数: 587 | 42 想对大概200bp的oligo template做in vitro transcription
我的目的就是，对oligo进行突变，比如一个A，一个C， 然后做IVT， 然后就简单的
nanodrop测量RNA yield；来观察A/C对transcription的影响
protocol上说对于short template，IVT可能有点困难。我想请问为啥呢？因为
polymerase很大，polymerase initiation需要一定的空间距离？而200bp太短？
我的目标并不是追求max yield，我的目标是比较A和C的yield区别。但是，因为200bp
长度太短，我不知道用200bp做IVT比较yield，是否严谨？ |
|
m******u
发帖数: 12400 | 43 RNA polymerase一般需要双链template---assume大家用的是T7或SP6的polymerase。你
200nt template指的是单链模板么？至少promoter区要双链的。
而PCR产物则是双链的，你的问题会不会是这个原因。
发信人: reallyloveme (ray), 信区: Biology
标 题: Re: in vitro transcription for short template
发信站: BBS 未名空间站 (Tue Jun 20 12:46:24 2017, 美东)
很感激
我做了200nt oligo为template的IVT，压根不work
而PCR product作为template还可以
我不知道是我技术的问题，还是oligo本来就很难work呢？ |
|
r**********e
发帖数: 587 | 44 DNA template做in vitro transcription，做完后，用bio-spin column去除single
nucleotide；
我这里是要比较SNP对于IVT的影响
那么接下来如何定量呢？用nanodrop定量，可以吗？
RNA浓度很高（IVT一般都产生大量RNA），nanodrop定量大概500ng/ul
总担心nanodrop不够accurate；但对于这么大量的IVT RNA， 难道用qPCR呢？我们做一
般total RNA 的qPCR，是要gapdh这样的control的，但IVT RNA，就一种sequence。。
。 |
|
m*****d
发帖数: 566 | 45 照现在的制药业状况，大药厂的研发部门，是做in vivo前景好些，还是做in vitro前
景好些? 我知道discovery整体就不乐观，但是我只有discovery方面的background.
面临选择，请大牛给说说? |
|
h********e
发帖数: 2823 | 46 Source:
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/H
How to Market Your Device
Please note: as of October 1, 2002, FDA charges fees for review of Premarket
Notification 510(k)s and Premarket Approvals
Introduction
Three Steps to Obtaining Marketing Clearance from CDRH
Classify Your Device
Selecting the Appropriate Marketing Application
Other Requirements Besides Marketing Clearance
In Vitro Diagnostic Devices
Introduction
One of the most difficult aspects of getting a medical... 阅读全帖 |
|
s**u
发帖数: 9035 | 47 Dr. Samuel M Cohen(Chairman of Department of Pathology, UNMC) wrote a letter to "Toxicological Science" to criticize a misleading from Chengfeng Yang(Michigan State University)lab's a recent paper published in this journal, see below:
Reply to “Reversal and Prevention of Arsenic-Induced Human Bronchial
Epithelial Cell Malignant Transformation by MicroRNA-200b”
1. Samuel M. Cohen1
+ Author Affiliations
1. Department of Pathology and Microbiology, University of Nebraska
Medical Center, Omaha... 阅读全帖 |
|
i****x
发帖数: 17565 | 48 你那个国内烂校学报的”科研“结论跟以下所有结论矛盾的时候，你说我们该信谁呢？
In 2006 a large Danish group's study about the connection between mobile
phone use and cancer incidence was published. It followed over 420,000
Danish citizens for 20 years and showed no increased risk of cancer.[21] A
2011 follow-up confirmed these findings.[22]
The following studies of long time exposure have been published:
The 13 nation INTERPHONE project – the largest study of its kind ever
undertaken – was published in 2011 and did not find a solid li... 阅读全帖 |
|
M*****e
发帖数: 279 | 49 McKusick-Nathans Institute of Genetic Medicine > News and Events >
Newsletter Stories > 2010_12
Fertile Ground: What 2010 Nobel Laureate Robert Edwards Did On His Summer
Vacation (in 1965)
October 2010- In 1965, Robert Edwards spent six weeks at the Johns Hopkins
University School of Medicine working as a visiting fellow with Howard W.
Jones Jr., M.D., then head of the crytogenetics laboratory, trying to
fertilize human eggs in a laboratory test tube. They failed. Or so they
thought when they pu... 阅读全帖 |
|
y*****h
发帖数: 166 | 50 Position level – Research Associate III or Senior Research Associate –
will be determined based on candidate’s background and experience.
Responsibilities and Duties
Responsible for the set up and performance of both in vitro and in vivo
experiments. The candidate will set up and perform in vitro experiments
using purified primary cells from human blood or mouse tissue. The candidate
will be involved in complex in vitro experiments requiring high technical
skills and precise planning. Addition... 阅读全帖 |
|
