n*******e
发帖数: 27 | 1 i have only used RNaseH, RNaseT1 for cDNA stuff. and i donno
its function in mammalian.
let us see this one
Mol Cell Biol 1999 Dec;19(12):8361-71
in S.C., RNase H was found to remove RNA primer from Okazaki frag.
Biochemistry 1999 Nov 16;38(46):15097-103
for HIV and other retrovirus, RNase H function is essential for
activity of the RT.
J Biol Chem 1999 Oct 1;274(40):28270-8
Human RNase H was cloned and found to cleave DNA:RNA substrate,
then what function:)? antibody template switch? DNA replic |
|
M****e
发帖数: 70 | 2 come on, show time, folks. so should we all email assassin back and corrupt
his mail box? i won't discuss all of the questions listed by assasin. actually,
to read papers on Cell is always headache, particularly manuscripts by
biochemistry/cell biology people.
Fig2A shows that purified separase can utilize in vitro translated(IVT) hSCC1
as well as endogenous hSCC1 as substrate in an in vitro cleavage assay. I think
the IVT sample (in RRL?) should be purified first (avoid incomplete translation), |
|
j*******e
发帖数: 26 | 3 Science (2003) 299: 223-226
“These caspases cleave many intracellular substrates, ultimately leading to
cell death.”
Why pathway study is important?
"it's importmant for sure. without knowing the central death pathway, how
would people study the regulation of the pathway? Usually signalling pathway
are
conserved between species, so if we know the pathway, we can manipulate
apoptosis. if we want cancer cell to die, we stimulate the death pathway. if
we want degenerating neurons survive, we can pr |
|
d****e
发帖数: 10 | 4 总体评价是：
1。 同义的cSNP可能导致蛋白质结构变化不是新idea。
2。没有证实C3435T导致的reduced codon usage(from 47% to 35%)直接引起蛋白质结
构变化。
3。提醒大家在自己的研究中不能忽视同义cSNP。
讨论会上，对这篇文章最大的质问就是该文发表的研究数据并没有证实文章标题所说的：
A silent polymorphism in the MDR1 gene changes substrate specificity.
原文一开始就说
"The functions of P-gp for all single polymorphism plasmids as well as for
wild-type MDR1, as
measured by intracellular accumulation or by efflux of fluorescent compounds
, were not distinguishable
under standard conditions. HeLa cells expressiing double |
|
m********h
发帖数: 14 | 5 究竟有多少个药物作用靶点？
2007-02-10 15:26:03 / 个人分类：药物研发
究竟有多少个药物作用靶点?
推荐一篇很棒的review文章
什么是药物作用靶点？究竟有多少个药物作用靶点？这篇文章根据列出的标准，估计了
现有的药物靶点总数。
From: astnt的blog http://astnt.myresearchspace.cn
估算的靶点分类有以下几种：
* Enzymes (Table 1)
* Substrates, metabolites and proteins (Table 2)
* Receptors (Table 3)
* Ion channels (Table 4)
* Transport proteins (Table 5)
* DNA/RNA and the ribosome (Table 6)
* Targets of monoclonal antibodies (Table 7)
* Various physicochemical mechanisms (Table 8)
|
|
d*****r
发帖数: 2583 | 6 ☆─────────────────────────────────────☆
ppri (小地主) 于 (Wed Feb 4 14:20:37 2009) 提到:
测 O2 consumption 的时候，在substrate 量足够情况下，一加入ADP，反映就停止了
。可能是什么原因?
☆─────────────────────────────────────☆
hellozero (hellozero) 于 (Wed Feb 4 16:14:52 2009) 提到:
More detailed info please. Is it in the isolated mitochondria?
Are you using Clark-type electrode or the MitoXpress fluorescent dye?
☆─────────────────────────────────────☆
ppri (小地主) 于 (Wed Feb 4 16:18:17 2009) 提到:
isolated mitochondria
Clar |
|
k******g
发帖数: 38 | 7 这个一作是一个人,一期science,2篇research article
Identification of SCF ubiquitin ligase substrates by global protein
stability profiling.
Yen HC, Elledge SJ.
Science. 2008 Nov 7;322(5903):923-9.PMID: 18988848 [PubMed - indexed for
MEDLINE]Related articlesFree article
Global protein stability profiling in mammalian cells.
Yen HC, Xu Q, Chou DM, Zhao Z, Elledge SJ.
Science. 2008 Nov 7;322(5903):918-23. |
|
s******y
发帖数: 28562 | 8 Depends on how small the amount is,
you can use either organic solvent HPLC or Mass Spec
I don't understand why you want to use RT-PCR? Can RT enzyme use DNA as
substrate?
Why don't you use a regular q-PCR? |
|
s******y
发帖数: 28562 | 9 You should use Tdtomato, because it is bright and because it avoid most of
the intrinsic fluorescence (which is kind of green).
I won't use luciferase because it is hard to get the substrate to penetrate
the cell. |
|
r****r
发帖数: 379 | 10 one paper said that you can express an enzyme which can produce the
substrates
penetrate |
|
a****a
发帖数: 83 | 11 求文：
Detection of Enzymatic Activity of Transfer RNA Modification Enzymes Using
Radiolabeled tRNA Substrates
谢谢。email:c****[email protected] |
|
f****y
发帖数: 104 | 12 Note the first author:
Energy-dependent regulation of cell structure by AMP-activated protein
kinase.
Lee JH, Koh H, Kim M, Kim Y, Lee SY, Karess RE, Lee SH, Shong M, Kim JM, Kim
J, Chung J.
(How true is this paper, you may see this to get some clue:
Myosin light chains are not a physiological substrate of AMPK in the control
of cell structure changes.
Bultot L, Horman S, Neumann D, Walsh MP, Hue L, Rider MH.
FEBS Lett. 2009 Jan 5;583(1):25-8)
Sestrin as a feedback inhibitor of TOR that prevents |
|
e********n
发帖数: 113 | 13 Link:
http://www.sciencemag.org/cgi/content/abstract/272/5268/1655
Please send to a******[email protected]
Science 14 June 1996:
Vol. 272. no. 5268, pp. 1655 - 1658
DOI: 10.1126/science.272.5268.1655
Prev | Table of Contents | Next
REPORTS
Enzymatic Synthesis of a Quorum-Sensing Autoinducer Through Use of Defined
Substrates
Margret I. Moré, L. David Finger, Joel L. Stryker, Clay Fuqua, Anatol
Eberhard, Stephen C. Winans * |
|
z*******a
发帖数: 175 | 14 I have been trying to show protein interaction between a kinase and its
predicted substrate by regular co-
immunoprecipitation. but failed twice already.
boss suggested crosslink co-IP again and again, very nicely though. I heard
it is almost impossible to publish
protein interaction data coming out from crosslink co-IP.
every Daxia,
Anybody see any published paper having crosslink co-IP data in it? If you do
, please forward me the basic info
( journal and author or title). Thanks a lot.
any ot |
|
|
l*******d
发帖数: 101 | 16 目光呆滞眼半盲，
颈项僵直臂酸胀。
双脚麻木屁屁痛，
精神渐癫狂。
尤其是看到某文中描述所用substrate的长度，写978bp 9次；写977bp 9次。几位灌水
大哥，好歹商量商量，拿个主意
嘛！ |
|
w****8
发帖数: 36 | 17 如果是什么enzyme的话，有时候加点substrate也是有用的。我们这边有个人做一个管
DNA damage的酶，一开始怎么做都做不出来，后来加了点DNA就出来了。 |
|
c*******2
发帖数: 25 | 18 J Biochem. 2009 Nov;146(5):659-65. Epub 2009 Jul 23.
Mycolyltransferase from Mycobacterium leprae excludes mycolate-containing
glycolipid substrates.
Nakao H, Matsunaga I, Morita D, Aboshi T, Harada T, Nakagawa Y, Mori N,
Sugita M.
Laboratory of Cell Regulation, Institute for Virus Research, Kyoto
University, Kyoto 606-8502, Japan.
E******************[email protected]
Thank you very much! |
|
s******y
发帖数: 28562 | 19 Because on soft substrates the cells don't want to form adhesions with it.
why |
|
m***n
发帖数: 337 | 20 GSK3{beta} mediates high glucose-induced ubiquitination and proteasome
degradation of insulin receptor substrate-1
PMID: 20466847
if you could access this paper, could you please send a copy to my email:
m******[email protected]. thank you very much |
|
k******e
发帖数: 8870 | 21 1. Theoretical Studies on Interaction Mode Between
Human 2-Amino 3-Carboxymuconate
6-Semialdehyde Decarboxylase and Substrate and
Inhibitor
Source: CHEMICAL JOURNAL OF CHINESE UNIVERSITIESCHINESE
Volume: 29 Issue: 12 Pages: 2398-2402
2. Bioisosteric replacement of molecular scaffolds: From
natural products to synthetic compounds
Source: NATURAL PRODUCT COMMUNICATIONS Volume: 3 Issue:
8 Pages: 1355-1360 Published: 2008
thanks a lot! |
|
z*********4
发帖数: 146 | 22 Theoretical Studies on Interaction Mode Between
Human 2-Amino 3-Carboxymuconate
6-Semialdehyde Decarboxylase and Substrate and
Inhibitor
Source: CHEMICAL JOURNAL OF CHINESE UNIVERSITIESCHINESE
Volume: 29 Issue: 12 Pages: 2398-2402
http://www.sendspace.com/file/c1xa5m |
|
f******5
发帖数: 7 | 23 *Job Description:*
A post-doctoral position is available in Dr. Sing Yian Chew’s laboratory http://www.ntu.edu.sg/home/sychew at the School of Chemical & Biomedical Engineering, Nanyang Technological University, Singapore, to study the effects of nanotopography on cell-substrate interactions and stem cell differentiation.
*Job requirements:*
Applicants should have at least a Ph.D. and skilled in cell biology and
stem cell differentiation and possess relevant experimental skills.
Applicants wit |
|
|
p*3
发帖数: 45 | 25 Caspase-3
Dixit VM.
Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable
protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell.
1995 Jun 2;81(5):801-9.
Miller, D. K.
Identification and inhibition of the ICE/CED-3 protease necessary for
mammalian apoptosis. Nature 376: 37-43, 1995. [PubMed: 7596430]
Caspase-8
Dixit VM.
FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the
CD95 (Fas/APO-1) death--inducing signaling complex. Cell. 1996 Jun |
|
v*********t
发帖数: 9 | 26 谢谢有师兄师姐能帮我！ 谢谢 ：）
Adjustment of receptor-binding and neuraminidase substrate specificties in
avian–human reassortant influenza viruses
Yulia Shtyrya1 Contact Information, Larisa Mochalova1, 2 Contact Information
, Galina Voznova1, Irina Rudneva3, Aleksandr Shilov3, Nikolai Kaverin3
Contact Information and Nicolai Bovin1 Contact Information
Glycoconjugate Journal
Volume 26, Number 1 / janvier 2009
http://www.springerlink.com.gate2.inist.fr/content/n8600j6n6303451t/
谢谢邮件联系： a**********[email protected] |
|
s******y
发帖数: 28562 | 27 Supersignal ELISA Femto
or Supersignal West FEMTO
both from Pierce Chemical (now under Fisher) |
|
|
m***c
发帖数: 637 | 29 招聘：中国联合利华 金属表面处理 或 生物材料研究助理。欢迎海外留学人士。主要
针对上海地区，但可以帮助寻找其它城市工作。请注明要求。
If you are interested, please email your CV to d****[email protected] with the
position you are applying as the
title.
助理研发专家(金属表面处理)
Job Responsibility:
1. Design and preparation of novel micro/nano structure, esp. on metal
substrates.
2. Surface modification skills through either chemical or physical
methodologies.
3. Develop product prototypes and make evaluations relevant to real
applications.
4. Raw materials scout and evaluation. |
|
a****d
发帖数: 1919 | 30 somethings you can take a look,
fruitless Splicing Specifies Male Courtship Behavior in Drosophila
Ebru Demir and Barry J. DicksonGo To Corresponding Author
The Sex-Determination Genes fruitless and doublesex Specify a Neural
Substrate Required for Courtship Song
Elizabeth J. Rideout1, Jean-Christophe Billeter1, 2 and Stephen F. Goodwin1,
Go To Corresponding Author |
|
y******8
发帖数: 1764 | 31 Good caculation.
The best way is isolating the DNA ploymerase, and examing the activity using
dNTA as substrates. Without enzymatic evidence, this is nonsense. |
|
a***e
发帖数: 1010 | 32 if the 5'-end of your ssRNA is -OH, you can add a Pi by T4 PNK.
although your substrate is gamma-Pi from ATP, the product is alpha-Pi. |
|
c**a
发帖数: 94 | 33 遇到高手了,讨教一下. 我用的是HDAC6, 从 Enzo 买的, 拿来做tubulin deAc, 这个在
2002的paper
中就show过了, 但是我做死都做不出来. 到底是买的protein失活了,还是反应需要特殊
的条件? 也
试过用Hela nuclear extract 加purified Histone, 也做不出来. 请高手明示. 另外,
你说的 TFA
substrate是什么?
most
though. |
|
v***a
发帖数: 1242 | 34 谢谢！前面也有位同学说用质谱。这个没有做过，正在研究中。
能请教一下p-y-100是哪家的抗体？
其实我不需要知道确切的磷酸化位点，只是想知道这个kinase让substrate的Tyr、Ser
、Thr磷酸化是增强还是减弱。
4G10 |
|
H******y
发帖数: 183 | 35 我们用SuperSignal® West Femto Maximum Sensitivity Substrate， 信号很强
，基本没有什么东西出不来的。原来用弱的试剂的时候，曾经过夜 |
|
s***m
发帖数: 6197 | 36 内行的能不能给解释一下为什么这个文章能在nature发呢？
感觉相关的结构也解了很多
这个分辨率也不算是很高
是不是就是因为有substrate在结构里呢？
谢谢！ |
|
s*********s
发帖数: 110 | 37 235425 Caspase-3 Substrate II, Fluorogenic
calbiochem
另外可以做western confirm, 比较好的caspase 3 antibody用cell siganling的9662 |
|
h****r
发帖数: 152 | 38 Try this. Never fail me
SuperSignal West Femto Maximum Sensitivity Substrate |
|
f*******h
发帖数: 38 | 39 吴号Michael Karin强强联手
搞结构灌水灌的真快
Crystal structure of inhibitor of κB kinase β
Guozhou Xu, Yu-Chih Lo, Qiubai Li, Gennaro Napolitano, Xuefeng Wu, Xuliang Jiang, Michel Dreano,
Michael Karin & Hao Wu
Department of Biochemistry, Weill Cornell Medical College, New York, New York 10021, USA
Guozhou Xu, Yu-Chih Lo, Qiubai Li & Hao Wu
Department of Pharmacology, University of California at San Diego, La Jolla, California 92093, USA
Gennaro Napolitano, Xuefeng Wu & Michael Karin
Nature (2011) doi:1... 阅读全帖 |
|
h*******o
发帖数: 4884 | 40 western 信号强化３步流
１）转膜完了用Ｑentix强化一把
２）Ｄevelop的时候用femto substrates
３）用传统胶片取代ＣＣＤ的电子系统
都不行的话，换抗体吧 |
|
z********i
发帖数: 610 | 41 怎么没有人讨论这篇文章了？
看了一下这几篇文章，很有可能NEDD4不是PTEN的E3，但是那篇CELL paper的方法和数
据又没有什么问题?jiang是从xiaodong wang实验室出来的,他从in vitro assay的建立
，到寻找组分的方法，和 xiaodong wang的一摸一样，但是为什么最后找到的蛋白为什
么不对呢？我感觉是in vitro assay的问题，虽然E3有很好的substrate specifictiy
，但是overexpressed 或者是in vitro assay的时候就不是这样了，过表达的情况下就
没有底物特异性了，所以在他们的assay 中能看到PTEN ubiquitination.后面可能选择
了符合预期的实验结果。一些相互作用和WB，多做几次总能出现一次想要的。junjie
chen的文章提到nedd4和pten没有相互作用，可以解释为实验条件不一样，比如 buffer
的离子强度。
我也是做生化的，对一些生化的结果总是很困惑。比如做个screening，曾经做过
kinase and phosphatase,发现一些kinase/... 阅读全帖 |
|
A******d
发帖数: 571 | 42 http://www.sciencemag.org/gca?sendit.y=7&gca=sci%3B332%2F6035%2
#
The mTOR-Regulated Phosphoproteome Reveals a Mechanism of mTORC1-Mediated
Inhibition of Growth Factor Signaling
* Peggy P. Hsu,
* Seong A. Kang,
* Jonathan Rameseder,
* Yi Zhang,
* Kathleen A. Ottina,
* Daniel Lim,
* Timothy R. Peterson,
* Yongmun Choi,
* Nathanael S. Gray,
* Michael B. Yaffe,
* Jarrod A. Marto,
* and David M. Sabatini
Science 10 June 2011: 1317-1322.
# Abstract ... 阅读全帖 |
|
w********h
发帖数: 12367 | 43 看文章不能老盯杂志的影响因子，
这三篇文章都超过100次，还不错啦。
Title: Comparative study of the use of poly(glycolic acid), calcium alginate
and pluronics in the engineering of autologous porcine cartilage
Author(s): Cao YL, Rodriguez A, Vacanti M, et al.
Source: JOURNAL OF BIOMATERIALS SCIENCE-POLYMER EDITION Volume: 9 Issue:
5 Pages: 475-487 Published: 1998
Times Cited: 149
Title: Tissue engineered neocartilage using plasma derived polymer
substrates and chondrocytes
Author(s): Sims CD, Butler PEM, Cao YL, et al... 阅读全帖 |
|
k***g
发帖数: 4904 | 44 我的substrate不是平的，用phase contrast应该也没法分辨。谢谢楼上几位！ |
|
S*****3
发帖数: 720 | 45 “中国百篇最具影响优秀国际学术论文” 评选所选论文代表了我国科技论文发展的最
高水平。论文源为前一年被SCI（科学引文索引）收录的中国论文。评选综合考虑发表
论文的期刊水平（影响因子和单篇引用次数）、论文类型、热点论文、论文的合作强度
、参考文献数和论文的完整性等方面。
其中生物类包括：
论文题目: Acute promyelocytic leukemia: from highly fatal to highly
curable
论文作者: Wang, Zhen-Yi; Chen, Zhu
所属机构: 上海交通大学医学院瑞金医院
来源期刊: BLOOD, 2008, 111(5):2505-2515
被引次数: 29
作者简介：
陈竺
卫生部部长，中国科学院院士，博士生导师
白血病系统生物学研究组组长
论文题目: Sorting of small RNAs into Arabidopsis argonaute complexes
is directed by the 5 ' terminal nucleotide
论... 阅读全帖 |
|
c********b
发帖数: 363 | 46 做EMSA时Kd差不多。有个朋友拿去做kinase substrate,效果不错。别的就没经验了。 |
|
s****t
发帖数: 461 | 47 30 years ago, some famous scientists faced the similar problem to yours.
Later, they solved the problem and their work got them Nobel price. Yes, I
am talking about RNA self splicing.
Unfortunately, you probably can't chemically synthesize your protein in full
-length. So it's almost impossible to get a truly clean system to demonstate
it.
Here are some suggestions:
1. Try in-vitro translation system to see if it's still the case.
2. If you can map the cleavage site, you can synthesize some pept... 阅读全帖 |
|
x********u
发帖数: 430 | 48 You need to get the initial velocity first before you can get kcat or Km.
Determine if the enzyme kinetics can be described by Michealis-Menten
eaquation or whatever inhibition equation,then get Km.Initial velocity can
be calculated by linear regression form the time serial data of product
formation or substrate consumption. |
|
p*****n
发帖数: 981 | 49 you need to determine the kcat and km for both A and B.
that is the only accurate way to tell the substrate specificity.
those two experiments you have done are rough, not very convincing.
1/ |
|
w******e
发帖数: 1187 | 50 用clear plate能有signal吗？刚order出去opaque plate，想
先用clear plate试试，有戏吗？ |
|
