E*******2
发帖数: 131 | 1 我正在做ChIP-qPCR， anti acetyl Histone H3 and H4, 将来也会考虑做anti
methylation histones，现在遇到的问题
是，找不到合适的control gene primers. Millipore 公司提供的chip kits里用的
GAPDH。 但是我们领域有几篇文章是用
的beta globin和beta tubulin。我的老板说用我们领域已发表的。然后我查了一下他
们用的primer sequences，有意思的
是他们用的是小鼠，但是primer sequences和大鼠的基因是完全匹配的，而和小鼠有1
－2个碱基不匹配。并且beta
globin的primer在大鼠基因中扩增的是hemoglobin epsilone2，而在小鼠中最可能扩增
的是hemoglobin y， beta－like
embryonic chain。 我的问题是，如果我选择beta globin做为我的control gene，我
是否应该重新设计primer，还是说
我可以直接用人家发表的primer sequence？但是他们用的是sybr |
|
c*********r
发帖数: 1312 | 2 Anti-acetylated tubulin antibody staining and phalloidin staining in the
starlet sea anemone nematostella vectensis.
Genikhovich, G.
Cold Spring Harbor protocols
Volume 4
Issue 9
Year 2009
ISSN/ISBN 15596095
都有哪些学校订了这个杂志啊，好多看着有用的protocol就是下不了全文。。。
多些了！ |
|
g*******f
发帖数: 427 | 3 最近对这个histone acetylation and deacetylation 调控基因表达挺有兴趣，就这个
HDAC好像很多文章的结论 contradictive . 不知道该怎么follow 了，是不是这方面很
难研究啊，还请有做这方面的牛兄弟给点意见。
刚发现澳大利亚一个组的一篇JBC文章，看摘要觉得挺有趣的，可是看全文发现被撤销了
VOLUME 284 (2009) PAGES 35101–35112
DOI 10.1074/jbc.A209.061903
Histone deacetylase-1 is enriched at the platelet-derived
growth factor-D promoter in response to interleukin-1
and forms a cytokine-inducible gene-silencing complex
with NF-B p65 and interferon regulatory factor-1.
Mary Y. Liu and Levon M. Khachigian
This art |
|
R****n
发帖数: 708 | 4 epigenetic有几个大分支，我们做的是DNA methylation detection.其他的还有
histone acetylation/methylation，别的我也不知道。DNA methylation和microarray
/next generation sequencing 结合比较紧密.可以做为癌症早期检测的评估手段。我
现在对methylation调控机理挺感兴趣，但是我们实验室不玩cell culture.大牛来讲讲
吧 |
|
C******s
发帖数: 1282 | 5 帮忙找2篇文章吧。烂学校的图书馆找不到啊。多谢多谢。请发到 w***[email protected]
1, The HTLV-1 Tax protein: Revealing mechanisms of transcriptional
activation through histone acetylation and nucleosome disassembly
Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms
Volume 1799, Issues 3-4, March-April 2010, Pages 266-274
2, Chapter 7 Nuclear Receptor Repression: Regulatory Mechanisms and
Physiological Implications
Progress in Molecular Biology and Translational Science
Volume 87, 2009, Pages 235-259 |
|
n********k
发帖数: 2818 | 6 Altered histone acetylation is associated with age-dependent memory
impairment in mice.
Peleg S, Sananbenesi F, Zovoilis A, Burkhardt S, Bahari-Javan S, Agis-Balboa
RC, Cota P, Wittnam JL, Gogol-
Doering A, Opitz L, Salinas-Riester G, Dettenhofer M, Kang H, Farinelli L,
Chen W, Fischer A.
Science. 2010 May 7;328(5979):753-6. Erratum in: Science. 2010 Jun 25;328(
5986):1634.
Recovery of learning and memory is associated with chromatin remodelling.
Fischer A, Sananbenesi F, Wang X, Dobbin M, Tsai |
|
n***w
发帖数: 2405 | 7 我们都知道beta-oxidation可以提供Acetyl-CoA和electron carriers。。。
我的问题是。。。beta-oxidation产生的carrier进入ETC。。。也是从complex I 传的
吗？还是说可以skip complex I?
谢谢。。。。 |
|
c**a
发帖数: 94 | 8 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. |
|
c**i
发帖数: 6973 | 9 Peter Landers, New Breast Cancer Drug Found Deep in the Sea. Wall Street
Journal, Jan. 4, 2010.
http://online.wsj.com/article/SB1000142405274870411150457605977
("A study found it [Halaven] extended the life of patients with advanced
breast cancer by about two-and-1/2 months, to 13 months")
My comment:
(a) Eisai Co., Ltd. エーザイ株式会社
(a public company; headquarters Tokyo; established in 1941 by Toyoji NAITO
内藤 豊次 as Nohon Eisai Co., Ltd. 日本衛材（衛生材料の略。具体的には絆創膏
や包帯の事))
ja.wikipedia.org
(b) eribulin
htt... 阅读全帖 |
|
|
c**********e
发帖数: 694 | 11 CoA has UV absorption, you can use HPLC-PDA to measure or LCMS |
|
v*******a
发帖数: 759 | 12 will the endogenous NADH affect the assay?
ab87546.html |
|
W*******a
发帖数: 1769 | 13 你做小老板连973项目组长都不能当，自然科学基金也只能拿1，2个，还不能申请
自然基金重点项目。生物是花大钱的学科，你没有钱，就无法学术独立，跟别人的差距
只会越来越大，逆水行舟，不进则退。其实这里的关键是你对学术到底有多大热情，你
是打算未来10年，15年的时间追求学术发展，还是想回国找个位子过日子而已。我已经
说了，生物这行，在中国还想做到跟美国一样好或者接近的地方，fresh phd是没有可
能拿到位置的。你要是愿意去二线城市的大学，弄成独立PI还是不成问题的，关键是那
样你也做不好science。
此外这也是有很多例外的，比如复旦去年发了两篇science, protein acetylation
那个人，听说就是一个副教授，半独立的PI，一下功成名就了。不过没有postdoc复旦估计
都不会给你副教授职位。还有一个可能是中科院正研下面的副研，不独立，你要是做的特别
好，一作cell，那么到别处去某个独立PI, professor估计还是可能的。但是那些东西
有太多的不确定性。还比如清华总跟施一公，颜宁一起发cns的wang jiawei，虽然是个
高工职称，估计也是... 阅读全帖 |
|
n******u
发帖数: 418 | 14 对于acetylation，是第二个情况
其余的modification不了解 |
|
s**x
发帖数: 138 | 15 i am going to pull down acetyl-h3k9. is it necessary or required to use
Histone deacetylase inhibitor NAM+TSA??? |
|
k*****n
发帖数: 323 | 16 where is your negative control? |
|
D*a
发帖数: 6830 | 17 Reduction of hippocampal N-acetyl aspartate level in aged APP(Swe)/PS1(dE9)
transgenic mice is associated with degeneration of CA3 pyramidal neurons.
Xu W, Zhan Y, Huang W, Wang X, Zhang S, Lei H.
J Neurosci Res. 2010 Nov 1;88(14):3155-60.
收到。 |
|
z********i
发帖数: 610 | 18 最近用Anti-acetyl-Lysine做WB，发现背景很高，膜上总是有一些小黑点，怀疑是
blocking的问题。用BSA blocking会好一些吗？不知道版上有没有人用这个抗体,wb的
条件是什么？
谢了。
antibody: millipore and Cell signaling Mouse mAb
blocking: 5% milk
time: 1hr
washing time after 2nd antibody: 1hr |
|
g****e
发帖数: 137 | 19 the most classic way to do this is to use radioactive SAM or stable isotope
labeled methyl group donor. There are many pan-methylation antibody
available, but don't expect too much for them. Unlike acetylation and many
other PTM, methylation has mono, di and tri-methylation, that's why is
difficult to develop an antibody recognize all three types of methylation. |
|
|
q****2
发帖数: 208 | 21 做个H3的chip，可能那个地方是nucleasome free region。 到时候normalize一下，那
个地方估计还是相对高的。 而且对的，多用几对primer 有时候primer的效率影响结果
也很大 |
|
|
c****1
发帖数: 1095 | 23 请教做乙酰化的专家：
1. 通常的乙酰化的抗体，都是用一个短肽做免疫原。比如，一个用来做K9ac抗体的短
肽，是4-15的序列，但是这个序列中，K9是ac修饰的，但是还有其他没有修饰的K，比
如K5，K12。我的问题是，这个抗体，是不是只能识别K9乙酰化，同时K5和K12没有修饰
的序列？这样一来的话，这个抗体，并没有识别所有的K9乙酰化的状态。好像还没有那
个paper提到这个情况。
2. 乙酰化修饰是否稳定？去乙酰化需要能量么？在RNAlater中解剖组织对乙酰化修饰
有没有什么影响？
非常感谢！！！ |
|
c****1
发帖数: 1095 | 24 非常感谢！！
抗体的问题，如果写paper，reviewer问到，改如何argue呢？对背景序列是否敏感，貌
似不好检测啊。我觉得，唯一的方法就是用同时应用不同公司的抗体，看是否有相同的
结果。
RNAlater是一种RNA稳定剂，主要用于RNA的提取，具体成分不详，作用机理就是让所有
蛋白变性。 |
|
T****O
发帖数: 407 | 25 1. Polyclonal Abs would be less specific to Ac-K9. Hypothetically, an Ab
could grab AA4-AA8 regardless antigens have Ac-K9 and Ac-K12 or not.
To get Ac-site-specific Abs you'll have to make them, affinity-purify and
confirm them with those peptides. |
|
c****1
发帖数: 1095 | 26 我觉得证明对ac-k9的特异性的最好的办法就是过表达k9的mutant， 然后用此抗体检测
。大家说呢？ |
|
A******y
发帖数: 2041 | 27 If that's what you want to do, you have to do both positive mutant (Lysine
to arginine) and negative mutant. Although, how many copies of histone H3
are there in human? I know for histone H4 there are more than 14 different
coding regions. |
|
l**********1
发帖数: 5204 | 28 if limited to Methyl transferase, Acetyl transferase,
Protein tyrosine phosphatase to perform a structural prediction of the putative ligands.
The modelling of the domains takes into account the different ligand
orientation when necessary as well as the orientation of the important
residues involved in binding.
Please go to
//adan-embl.ibmc.umh.es/
from
//www.embl.de/
and more pls go to
//mamba.bio.uci.edu/rt95.06.16/rt_1.html
from
//www.mcb.harvard.edu/hastings/dino.html |
|
z****u
发帖数: 1007 | 29 比如说，我能通过laser capture microscope分离出几百个stem cell, 几百个TA cell
。啥技术能比较他们的methylation, acetylation 等等的状态。 没做过，不懂。 |
|
z****u
发帖数: 1007 | 30 比如说，我能通过laser capture microscope分离出几百个stem cell, 几百个TA cell
。啥技术能比较他们的methylation, acetylation 等等的状态。 没做过，不懂。 |
|
l**********1
发帖数: 5204 | 31 Alleviating interactions.
Please go to AMPK(Sip2) Nature paper:
Functional dissection of lysine deacetylases reveals that HDAC1 and p300
regulate AMPK (2012)
link:
//www.ncbi.nlm.nih.gov/pubmed/22318606
its pp251
called both as
[synthetic lethal (negative/aggravating) and rescue (positive/alleviating)
interactions]
more reference:
Acetylation of yeast AMPK controls intrinsic aging independently of caloric
Cell 146: 969-
//www.ncbi.nlm.nih.gov/pubmed/21906795 |
|
b******y
发帖数: 627 | 32 Not complicated for the last strategy. free thiol from cys reacts with
maleimide very efficiently. tri-maleimide is cheap. you have to synthesize
peptide anyway.
Just a reminder: the N-termini of proteins in vivo (bacteria or mammalian
cell) are complicated. The majority of them have the met cleaved upon
translation. Also the majority of them are N-terminally acetylated
cotranslationally or post-translationally.
Be careful! Just want to remind you not to use wrong peptides for Ab
generation. |
|
s******y
发帖数: 28562 | 33 非常谢谢！ 我们准备订购的合成的多肽末端是有N-acetylation 的（所以不敢在
太小的公司那里订）， 而那个GST fusion万一真的需要做的话也准备在293T cell里面
制备
你知道得很多啊，谢谢。 |
|
o****e
发帖数: 1011 | 34 多长的peptide？（多少amino acid residue？）
仅是peptide合成的话，所谓"N-Terminal Acetylation"不过是多了一小步简单的
合成而已，还不至于跟“敢不敢”什么的联系起来吧。
mammalian |
|
j*p
发帖数: 411 | 35 1. 许多癌症的存活率之所以很低，很大一个原因，是相当一部分患者在确诊患癌症的
时候，已经是很晚期。相反，如果发现的早，那么按照现在的医学水平，还是有很多办
法可以治愈或者控制许多癌症，并以此延长患者的生命。
2. NGS以及其他技术（例如cell sorting, signal-cell seq/image）的发展，当然也
包括像TCGA等大型项目的实施，将在不久的将来对癌症的早期诊断获得突破，尽管目前
还存有许多实验上和分析上的技术问题。
3. 然而这些技术的发展对了解癌症机理的贡献，并以此提出新的治疗方案，或者发明
新的药物，短期内不会有太大的突破。不仅仅因为癌症，作为一个笼统的概念，是数百
个不同的疾病的总和，不同的癌症发病机理有可能会非常不同。这里牵涉到的问题非常
多，例如此前有同学说的，signaling pathway，gene and protein expression, post
transcriptional and translational modification等，也涉及到DNA/RNA
methylation，acetylation, histome m... 阅读全帖 |
|
b******y
发帖数: 627 | 36 The same holds true for many acetyltransferases although understudied. That
is to say, many acetyltransferases are acetylated around their active center
or substrate recognition pocket to be active. |
|
d****d
发帖数: 214 | 37 T2A编码的是一个peptide，可以连接两个蛋白质的coding sequence使其转录时为一个
mRNA但在翻译时分开成为两个独立的蛋白。
以你的问题为例子，你可以在T2A上游插入你感兴趣的蛋白质的coding sequence，最终
你的蛋白和puromycin resistant gene (Puromycin N-acetyl-tranferase)所编码的蛋
白会被翻译成两个蛋白。这种同时表达多个基因的策略同IRES策略相比，好处是上下游
蛋白的比例理论上讲是严格的1：1. |
|
A********o
发帖数: 1307 | 38 Deng X-D, Gu B, Li Y-J, Hu X-W, Guo J-C, Fei X-W (2012) The Roles of acyl-
CoA: Diacylglycerol Acyltransferase 2 Genes in the Biosynthesis of
Triacylglycerols by the Green Algae Chlamydomonas reinhardtii. Mol Plant 5:
945-947
Huerlimann R, Heimann K (2012) Comprehensive guide to acetyl-carboxylases in
algae. Critical Reviews in Biotechnology Ahead of Print: 1-17
Jansson C (2012) Metabolic Engineering of Cyanobacteria for Direct
Conversion of CO2 to Hydrocarbon Biofuels. Progress in Botany: Progr... 阅读全帖 |
|
A******y
发帖数: 2041 | 39 Unfortunately the substrate of SIRT6 is unlikely to be acetylated lysine, I
wounder what will it be. |
|
A******y
发帖数: 2041 | 40 Google SIRT5 and succinate etc. SIRT6 although has been reported to be a
deacetylase, but I think it is BS (don't worry no paper will be retracted,
just like all the class IIa HDAC paper). The activity is too low or none.
I think class IIa, IV, HDAC6 domain 1, and SIRT4, 6, and 7 substrates are
either not acetyl group or require special activation and highly context
dependent.
N.M. here is the link: http://www.ncbi.nlm.nih.gov/pubmed/22076378
P.S. I'm one of the reviewers of the SIRT5 paper. |
|
P*******e
发帖数: 27 | 41
SIRT5 is kind of special since it has tyr and Arg in the substrate site.
These residues can interact with the carboxylate of succinate. I am not sure
you can make such a generalization.
If SIRT6 works through regulation of transcription, probably it doesn't need
to be very active in terms of deacetylation. As long as H3K56Ac at the
promoter of target genes can be selectively deacetylated, the effect can be
amplified by the transcription process assuming the acetylation is slow. |
|
i*****g
发帖数: 11893 | 42 p53这个蛋白被做烂了
几乎每一个氨基酸都被摸过了
前几年，还有个尾巴lys的methylation(acetylation) 还发了一篇 N or S article，
你可以想象被研究到什么程度 |
|
a*****2
发帖数: 175 | 43 paraffin- or cryo- 都做过,cryo的信号好一些,最好切完一个星期以内作.
endogenous的biotin有kit可以inhibit,好像是vectorlab.不过,我用的是DIG labell的
probe.
降低背景,还需要作acetylation.这个步骤也可以替换成0.2NHCL处理10min.
最后最后,target gene的表达量是多少,如果太低,背景都会很高.
endogenous |
|
|
s*******2
发帖数: 598 | 45 See below 1997 paper
Biochem J. 1997 Apr 1;323 ( Pt 1):233-7.
Actin is cleaved during constitutive apoptosis.
Brown SB, Bailey K, Savill J.
SourceDivision of Renal and Inflammatory Disease, Department of Medicine,
University Hospital, Nottingham NG7 2UH, UK.
Abstract
Proteases play an important role in the programme of cell death by apoptosis
but little is known of the substrates cleaved, particularly in constitutive
models of this type of cell death. Neutrophils spontaneously undergo
apoptosis ... 阅读全帖 |
|
w**********s
发帖数: 8 | 46 Yuan is simply attacking the first fig of Lin et al Nature paper. And the
paper is really focused on AMPK and its acetylation regulation.
Yuan and Washington Post also admitted "he is questioning the first part.
and even if the first part is wrong, the second part of the paper can still
be right".
Lin should simply leave the genomics and statistics out of this paper.
What a pity. |
|
w**********s
发帖数: 8 | 47 Yuan is simply attacking the first fig of Lin et al Nature paper. And the
paper is really focused on AMPK and its acetylation regulation.
Yuan and Washington Post also admitted "he is questioning the first part.
and even if the first part is wrong, the second part of the paper can still
be right".
Lin should simply leave the genomics and statistics out of this paper.
What a pity. |
|
l****g
发帖数: 717 | 48 Article
Acetyl-L-carnitine suppresses apoptosis of thioredoxin 2-deficient DT40
cells.
Xiaoping Zhu, Eisuke F Sato, Yi Wang, Hajime Nakamura, Junji Yodoi, Masayasu
Inoue |
|
l****y
发帖数: 398 | 49 I have seen ser/thr acetylation on e.coli expressed recombinant histone once
. It was actually surprisingly abundant (~5%).
It could be modified by e.coli enzymes or in vitro by acetic acid or some
chemicals during sample processing. |
|
K****e
发帖数: 32 | 50 ser/thr acetylation 可能是由于处理过程中发生的酯化反应，如果是lysine上的乙酰
化呢？是否可以认为偏向于E. coli中酶的作用？
once |
|
