z***d
发帖数: 131 | 1 1.发现Overexpression of 蛋白A会导致gene B表达增高；
2.文献报道蛋白A是一个Non-histone chromatin remodeling protein，它能displace
HADC，从而增加acetyl-H3K9在另两个target genes(C and D)promoter regions处的结
合。
3.另有报道Gene B的表达受HDAC调控。
因此，我的Hypothesis是蛋白A也是通过相似的方式（displace HADC，增加acetyl-
H3K9在genes B promoter regions处的结合)来引起基因B的高表达。
可是今天的Acetyl-H3K9/K14 ChIP结果正好和假设完全相反：在蛋白A高表达的细胞中
，acetyl-H3K9在genes B promoter region和RPL 30 intron 2 (Positive control）
处的结合率显著降低。
疑问：1. 有acetyl-H3K9在promoter region处的结合率下降，但基因表达却开启的例
子吗？
2. 由于蛋白A本身又可以和Hi... 阅读全帖 |
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b**********8
发帖数: 349 | 2 2. 由于蛋白A本身又可以和Histone H1竞争与Linker DNA binding, 有无可能所见到的
acetyl-H3K9在genes B promoter region处的结合率降低是由于高表达的蛋白A本身取
代了endogenous H3 和acetylated-H3?
这个还真有可能。很多蛋白不但调节其他转录因子，他本身也可以作为转录因子直接参
与调控靶基因转录。要不再用A蛋白的抗体做个ChIP试试？另外，你可以做个western看
看pan-acetyl-H3K9的水平有没有降低。
楼下继续。 |
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g*********3
发帖数: 177 | 4 It is highly possible that there is no direct binding between protein A and
gene B sequence. If Protein A regulates another gene C and C regulate B,
ChIP doesn't work...
I don't quite understand what you mean by acetyl-H3K9在另两个target genes(C
and D)promoter regions处的结合。 Do you mean H3K9 acetylation level @ these
two promoters?
displace |
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A******y
发帖数: 2041 | 5 For number 1, if you are worry about reviewers, you have to do two things: 1
. Mass spec will tell you where the modification is. 2. For your gene of
interest, you can use different but well know histone acetylation or
methylation marker doing chromatin IP. Both require experience and special
training to do them right though. Even if you can find specific actylated
ab for the resiude, reviewers will ask for these two experiments for a good
journal. As for as I know, histone H3 K9 Chip is ve... 阅读全帖 |
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fe
发帖数: 286 | 6 求一批Nature's Bounty的acetyl L-carnitine，其他牌子的也可以，站内联系或email：
g********[email protected] |
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c**a
发帖数: 94 | 7 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。 |
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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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A******y
发帖数: 2041 | 9 What's your substrate? Are you using Enzo substrate? If your hela cell
extract didn't work, don't expect the HDAC6 to work.
Check your pH...should be 8
Do the incubation at room temp first.
HDAC6 is fragile...I will get them from BPSbiosciences.
Go buy a tube of Boc-Lys(ac)-AMC from Bachem or Anaspec and make the
substrate youself in DMSO...
watch your %DMSO...more than 1% in the reaction mix will not work (yea, I
know someone say they have 5%...sure)...
If you are trying to deacetylate isolat... 阅读全帖 |
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x***n
发帖数: 120 | 10 需要测定小鼠各种组织样品中的Acetyl CoA含量。
老板非常需要知道这个结果。
我搜了一下，没有什么头绪。有没有做过这个实验的同学，帮忙提点一下！
不胜感激！！ |
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m******t
发帖数: 109 | 11 i pulled down chromatin with anti-acetyl H3k9 antibody and normal IgG as
control. then i did q-PCR to pick up a DNA segment in a promoter. i found,
compared with normal IgG, the specific antibody enriched hundreds -over 1
thousand times of target DNA segment. does it make sense? |
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p******g
发帖数: 180 | 12 一般是用anti-H3，anti-acetyl H3k9 一起做，anti-H3作为control。 |
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a**********s
发帖数: 8 | 13 和我前一段时间做的结果接近。IgG大约为Input的0.01％，Acetyl H3k9为10％ |
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z***d
发帖数: 131 | 14
另外，你可以做个western看
做过Western, pan-acetyl-H3K9没有减低，反而modestly increased. |
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s******y
发帖数: 28562 | 15
取决于具体的抗体，有的是可以识别所有的acetyl-lysine，有的则对背景序列
也敏感
一般比较稳定。
去乙酰化不需要能量，但是需要酶催化才能高效。
RNAlater具体是什么成分？如果不是碱性很强， 应该问题不大。 |
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c****1
发帖数: 1095 | 16 多谢回答!
1.Mass Spec不错，不仅可以知道哪些位点被修饰了，而且可以量化。但是，我的样品
是组织，同一个位点可能有多个修饰，Mass Spec可以分辨出来么？
2. ChIP貌似也是运用抗体，跟WB一样，也不能辨别它对背景序列的敏感程度吧？
3. 我听说，TSA可以人为的增加Acetylation的程度，有这回事么？
1
special
good |
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s******y
发帖数: 28562 | 17 抗体的问题很简单，你找公司去合成另外几个 peptide，在acetyl-Lysine
附近放进不同的氨基酸，用它们作为control来测试你的抗体。
关于你对另外一个人的问题：Mass Spec 完全可以知道是那个残基上的修饰 |
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s*****c
发帖数: 5202 | 18 Nature's Bounty Acetyl L-Carnitine?
Acetyl L-Carnitine
Acetyl L-Carnitine assists in the breakdown of fat in cells to produce
energy.* Scientific studies show that Acetyl L-Carnitine promotes brain
metabolism, memory and cognitive function, especially mild memory problems
associated with aging.* Acetyl L-Carnitine can be used as part of a healthy
aging program. Nature's Bounty Acetyl L-Carnitine comes in convenient, 250
mg, easy-to-swallow capsules. |
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f*1
发帖数: 837 | 19 https://en.wikipedia.org/wiki/Acetylcarnitine
ALCAR is an acetylated derivative of L-carnitine. During strenuous exercise,
a large portion of L-carnitine and unused acetyl-CoA are converted to ALCAR
and CoA inside mitochondria by carnitine O-acetyltransferase. ...
Excess acetyl-CoA causes more carbohydrates to be used for energy at the
expense of fatty acids. ...
ALCAR is also known to increase sperm motility, which describes the
ability of sperm to move vigorously. Since motility is among the m... 阅读全帖 |
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h**********r
发帖数: 671 | 20 做微生物的人说说：pyruvate还是会利用pdh产生acetyl-CoA,但是这是在mitochondria
里的，不能直接合成lipid。然后利用部分的TCA cycle转化成citric acid,这个可以进
入cytoplasm.在citric acid lyase作用下合成acetyl-CoA。TCA cycle可以部分的shut
down,甚至可以有reverse,保证大量的碳源进入到lipid的合成。同时有NADPH的产生。
前段时间炒的很火的IDH，其实和这个或许有联系。也许在cytoplasm有pdh bypass产生
acetyl-CoA，不是主要来源。acetyl-CoA产生manly CoA是合成脂肪酸的第一步，由ACC
催化完成。这个受很多调控，例如citric acid的诱导，和磷酸化调控（AMP, AMPK）。
pyruvate mitochondria carrier不是找到了一部分了么（science还是nature背靠背文
章）?好像knockout对lipid没啥太大的影响。 |
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d*****s
发帖数: 647 | 21 Nature. 2013 Nov 7;503(7474):146. doi: 10.1038/nature12727.
Retraction: Functional dissection of lysine deacetylases reveals that HDAC1
and p300 regulate AMPK.
Yu-yi Lin, Samara Kiihl, Yasir Suhail, Shang-Yun Liu, Yi-hsuan Chou, Zheng
Kuang, Jin-ying Lu, Chin Ni Khor, Chi-Long Lin, Joel S. Bader, Rafael
Irizarry & Jef D. Boeke
Nature 482, 251'255 (2012); doi:10.1038/nature10804
In response to a concern raised by a reader about inconsistencies in our
Letter between the results from the primary mi... 阅读全帖 |
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h********y
发帖数: 627 | 22 《Science》杂志同时刊发两篇复旦生物医学最新研究成果：
复旦大学发现蛋白质乙酰化修饰的新机制
代表性论文和专著[Selected Publications, * corresponding author(s)]
1. Shimin Zhao, Wei Xu, Wenqing Jiang, Wei Yu, Yan Lin, Tengfei Zhang, Jun
Yao, Pengyuan Yang, Hong Li, Yixue Li, Jiong Shi, Lunxiu Qin, Qunying Lei,
Yue Xiong, and Kun-Liang Guan. Protein acetylation plays an extensive role
in regulating cellular metabolism. 提交至 Science, accepted in principle.
2. Qijun Wang, Yakun Zhang, Hui Xiong,Yan Lin, Hong Li, Lu Xie, Wei Zhao,
YufengYao, Yue Xio... 阅读全帖 |
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s****a
发帖数: 1039 | 23 http://www.sciencemag.org/current.dtl
Regulation of Cellular Metabolism by Protein Lysine Acetylation
Shimin Zhao, Wei Xu, Wenqing Jiang, Wei Yu, Yan Lin, Tengfei Zhang, Jun Yao
, Li Zhou, Yaxue Zeng, Hong Li, Yixue Li, Jiong Shi, Wenlin An, Susan M.
Hancock, Fuchu He, Lunxiu Qin, Jason Chin, Pengyuan Yang, Xian Chen, Qunying
Lei, Yue Xiong, and Kun-Liang Guan
Science 19 February 2010: 1000-1004.
Regulation of enzymes by acetylation controls metabolic function in human
liver cells.
Acetylation o |
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A******y
发帖数: 2041 | 24 That's only if you still believe that SIRTs are deacetylase. Their
activities compare to HDACs (metal containing deacetylases) are at least ten
times lower and reversible. Could it be that acetylation is actually done
by HDAC and a SIRT just serve as an recognition motif. The same mistake has
been made for class IIa HDAC. Unfortunately I still see people repeating
the wrong information on their papers. As for specific residue differences,
two domain of HDAC6 has little differences in amino ... 阅读全帖 |
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A******y
发帖数: 2041 | 25 It really doesn't matter if it is chemical modification or biological
modification. Some scientist in the field believe a lot of acetylation
modification is through direct chemical transfer of Acetyl-CoA without the
help of HAT espeically in organelle enviroment favoring nucleophilic
addition.
As far as crotonyl-modification. My friesd published in a good journal
stating HDAC3 is the de-crotonylase...my lab results are exactly opposite.
HDAC1 and HDAC2 have more activities against cortonyl-ly... 阅读全帖 |
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a*******y
发帖数: 400 | 26 acety这种对减肥有没有用我不知道，这个好象是给老人吃的。。。
我都买普通的那种，也便宜啊哈哈
——————
乙酰左旋肉碱 Acetyl-L Carnitine
cetyl-L-Carnitine (ALC)中文名为乙酰左旋肉碱，是L-肉碱的一种天然形态。乙酰左
旋肉碱在细胞呼吸中担任着传递脂肪酸进入粒线体的传递工具。帮助脂肪酸进入粒线体
从而产生能量体ATP。乙酰左旋肉碱和L-肉碱都具有运输脂肪的能力。和L-肉碱不同的
是，乙酰左旋肉碱可以穿过血脑障壁，提供脑细胞足够的能量。
根据美国VITY维他命杂志报道:"口服乙酰左旋肉碱可以增加血液，脑髓中乙酰左旋肉碱
的含量。 乙酰左旋肉碱能够帮助改善老年老年期抑郁症，预防治疗痴呆症。 提高学习
力与记忆力。 保护脑细胞，提供脑细胞足够的能量和乙酰辅脢A(acetyl-CoA)，活化脑
细胞的讯息传导。 保护细胞膜的稳定性。 提高神经生长因子的合成。对中枢神经和周
围神经元的生长、发育、正常状态的维持、损伤后的保护和轴突的有效再生都有着重要
作用。" |
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g********n
发帖数: 1015 | 27 左旋肉碱有2种， L-Carnitine 和 Acetyl-L-Carnitine. 有什么区别呢？
我去年底买的L-Carnitine ， 吃了2周，对boost体力的确有很大帮助。 通常要练1-2
个月才能达到的，1-2周就可以了。 再比如我昨天吃了一粒500mg和一粒200mg绿茶去跑
步， 跑步的速度时间都加大了10%， 总体就是增加20%。
但有个问题， 我去年吃了2周后（不是天天吃，就是运动前吃）， 觉得有忽然走神的
情况。停了以后就没有了。 今年3-4月， 吃了4-5次， 又出现了。
不知道有没有人有类似状况。
我上个月吃的HCG里也有左旋， 但是Acetyl-L-Carnitine， 却没有任何反应。 也可能
是剂量小。 |
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g********n
发帖数: 1015 | 28 左旋肉碱有2种， L-Carnitine 和 Acetyl-L-Carnitine. 有什么区别呢？
我去年底买的L-Carnitine ， 吃了2周，对boost体力的确有很大帮助。 通常要练1-2
个月才能达到的，1-2周就可以了。 再比如我昨天吃了一粒500mg和一粒200mg绿茶去跑
步， 跑步的速度时间都加大了10%， 总体就是增加20%。
但有个问题， 我去年吃了2周后（不是天天吃，就是运动前吃）， 觉得有忽然走神的
情况。停了以后就没有了。 今年3-4月， 吃了4-5次， 又出现了。
不知道有没有人有类似状况。
我上个月吃的HCG里也有左旋， 但是Acetyl-L-Carnitine， 却没有任何反应。 也可能
是剂量小。 |
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m******i
发帖数: 9479 | 29 我做research的时候看到有人吃带acetyl的更管用。
我买的是带acetyl的，反正就是没啥用= = 。不带的也没啥用。。 |
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b****i
发帖数: 11322 | 30 我来帮你找吧。你以为肝分解一步就直接排了？呵呵
from wiki on Ethanol metabolism ：
The first three steps of the reaction pathways lead from ethanol to
acetaldehyde to acetic acid to acetyl-CoA. Once acetyl-CoA is formed, it is
free to enter directly into the citric acid cycle. |
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u*********1
发帖数: 2518 | 31 Once a jolly pyruvate enters the matrix
Of a mitochondrion, so they say,
A decarboxylating, complex dehydrogenase
Converts it to acetyl coenzyme A
Waltz around the cycle,
Waltz around the cycle,
Waltz around the TCA cycle today,
A decarboxylating, complex dehydrogenase
Turns pyruvate to acetyl CoA
Oxaloacetate looking for a partner
Thinks "active acetate" looks OK,
Condensing enzyme arranging a merger
Makes a new citrate, and kicks out CoA.
Waltz around the cycle,
Waltz around the cycle,
Waltz a... 阅读全帖 |
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u*********1
发帖数: 2518 | 32 Once a jolly pyruvate enters the matrix
Of a mitochondrion, so they say,
A decarboxylating, complex dehydrogenase
Converts it to acetyl coenzyme A
Waltz around the cycle,
Waltz around the cycle,
Waltz around the TCA cycle today,
A decarboxylating, complex dehydrogenase
Turns pyruvate to acetyl CoA
Oxaloacetate looking for a partner
Thinks "active acetate" looks OK,
Condensing enzyme arranging a merger
Makes a new citrate, and kicks out CoA.
Waltz around the cycle,
Waltz around the cycle,
Waltz a... 阅读全帖 |
|
n*********m
发帖数: 38 | 33 Look his publication
Conversion of D-ribulose 5-phosphate to D-xylulose 5-phosphate: new insights
from structural and biochemical studies on human RPE.
Liang W, Ouyang S, Shaw N, Joachimiak A, Zhang R, Liu ZJ.
FASEB J. 2011 Feb;25(2):497-504. Epub 2010 Oct 5.
PMID:
20923965
[PubMed - indexed for MEDLINE]
Related citations
2.
Structural basis for the inhibition of human 5,10-methenyltetrahydrofolate
synthetase by N10-substituted folate analogues.
Wu D, Li Y, Song G, Cheng C, Zhang R, Joac... 阅读全帖 |
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A******y
发帖数: 2041 | 34 Simplified version for acetylation is number 2. However, acetylated histone
also recruit activation factors and displace silencing factors. Usually
for all transcription number 1 combining with number 2 is correct, don't
forget the HAT/HDAC has to be regulated to know which genes to turn on and
off. |
|
E********y
发帖数: 1048 | 35 Sci Signal. 2012 Jul 3;5(231):pe29.
New targets for acetylation in autophagy.
Hamaï A, Codogno P.
Autophagy. 2012 Oct 1;8(10).
How does acetylation regulate autophagy?
Yi C, Yu L.
email:
a******[email protected]
Thanks a lot. |
|
|
d*********w
发帖数: 6 | 37 这个Lin YY 很高产啊，三年内三篇CNS。高度怀疑其他paper的可信度。。。那个Lu JY
好像是他老婆。
Functional dissection of lysine deacetylases reveals that HDAC1 and p300
regulate AMPK.
Lin YY, Kiihl S, Suhail Y, Liu SY, Chou YH, Kuang Z, Lu JY, Khor CN, Lin CL,
Bader JS, Irizarry R, Boeke JD.
Nature. 2012 Feb 8;482(7384):251-5. doi: 10.1038/nature10804.
Acetylation of yeast AMPK controls intrinsic aging independently of caloric
restriction.
Lu JY, Lin YY, Sheu JC, Wu JT, Lee FJ, Chen Y, Lin MI, Chiang FT, Tai TY,
Berger SL, Zhao Y, Ts... 阅读全帖 |
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A******y
发帖数: 2041 | 38 What are you talking about? They have activities, just no activity against
acetylated lysine. Sirtuin 5 and 6 used to be no activity against
acetylated lysine...their natural substrates known now. My interest is in
HDACs, let's hope I don't get scooped...too many projects not enough money
and able hands... |
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K**4
发帖数: 1015 | 39 Deacetylase specifically refers to removing the acetyl group, whereas
deacylase is more general in that it can remove R groups that are acetyl or
longer. Reaction wise they should just be the same. |
|
A******y
发帖数: 2041 | 40 That's because the ac-p52 antibody is horrible. Same thing for Ac-Hsp90 etc
. It is the best to I.P. the protein and do acetyl-lysine antibody...the
problem is that acetyl-lysine ab is horrible, too. |
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E**********t
发帖数: 56 | 41 Xuebiao Yao, Dasheng Li and Gang Pei recently wrote an essay to Nature
Review in MCB Which is entitled “In focus: molecular and cell biology
research in China”. Here is the link: http://www.nature.com/nrm/journal/v14/n9/full/nrm3638.html In this article, they discussed the past, present and future of molecular and cell biology research in China. One part which interested me is the key MCB discoveries in China. Although most of the work are nice and likely solid work, few of them are breakthrough... 阅读全帖 |
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r**t
发帖数: 64 | 42 Looks like the area of sirtuins research is a really mess. I never
understand why cells have to spend so much energy on acetylating proteins in
the first place and then removing the acetyl groups. |
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B******1
发帖数: 9094 | 43 Another way is to add acetyl chloride slowly to a stirred, cold methanol
solution. You can calculate the amount of acetyl chloride needed before hand.
But I don't know the solubility of HCl in MeOH. |
|
|
f**d
发帖数: 768 | 45 这是一本计算神经科学的优秀著作，全文拷贝这里（图和公式缺），有兴趣的同学可以
阅读
如需要，我可以分享PDF文件（－－仅供个人学习，无商业用途）
From Computer to Brain
William W. Lytton
From Computer to Brain
Foundations of Computational Neuroscience
Springer
William W. Lytton, M.D.
Associate Professor, State University of New York, Downstato, Brooklyn, NY
Visiting Associate Professor, University of Wisconsin, Madison
Visiting Associate Professor, Polytechnic University, Brooklyn, NY
Staff Neurologist., Kings County Hospital, Brooklyn, NY
In From Computer to Brain: ... 阅读全帖 |
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w********9
发帖数: 8613 | 46 以下是第19001到第20000的word families的开头：
ABAYA 0
ABAYAS 0
ABEAM 0
ABLATE 0
ABLATED 0
ABRACADABRA 0
ACETYL 0
ACIDOPHILUS 0
ACROMEGALY 0
ACROMEGALIA 0
acromegalic 0
ACTE 0
ACTES 0
ACTINIC 0
ACUPRESSURE 0
ACYL 0
ADAMANTINE 0
ADRENERGIC 0
ADRENERGICALLY 0
ADVERTORIAL 0
ADVERTORIALS 0
AEROGRAMME 0
AEROGRAM 0
AEROGRAMMES 0
AEROGRAMS 0
AFLATOXIN 0
AFLATOXINS 0
AITCH 0
AITCHES 0
ALENDRONATE 0
ALEPH 0
ALEWIFE 0
... 阅读全帖 |
|
发帖数: 1 | 47 褪黑激素(Melatonin)， 一场精心的骗局
以下文章详细叙述了褪黑激素的研究及其负面报道。其实，这种宣传，如果用脑子仔细
想一想，就能发现漏洞，这实际上是一场精心策划的骗局，又因为日本人的参与，使这
种产品更加危险。因为日本人自战后，就有了控制世界，灭绝对手的计划。来自日本的
一项研究专利表明，虾青素（ASTA）能够促进内源性褪黑素的分泌，并获得专利，专利
号为EP1283038，并且阐述因为褪黑素极易被机体的有两种氧氧化而失去作用，而虾青
素（ASTA）这种东西可以保护褪黑素不被氧化，因此用来调节时差。褪黑激素(
Melatonin)主要是由哺乳动物和人类的松果体产生的一种胺类激素。人的松果体是附着
于第三脑室后壁的、豆粒状大小的组织。也有报导哺乳动物的视网膜和副泪腺也能产生
少量的褪黑激素；某些变温动物的眼睛、脑部和皮肤(如青蛙)以及某些藻类也能合成褪
黑激素。想一想，就用脑子想一想，实验者如何去做这个人体实验？ 日本人是最残忍
的实验者，想一想二战的时候，为了测试人体的烫伤问题，直接就把美国战俘的手放在
了滚烫的热水里了。假设真的有这个实验，我相信这个实验也是异常残忍的一个... 阅读全帖 |
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发帖数: 42116 | 48 鸦片类止痛药芬太尼（fentanyl）遭滥用成瘾问题在美国越来越严重，日前过世的西洋
歌坛传奇王子（Prince）也是因为服用过量芬太尼致死。而根据美国缉毒署（DEA）调
查，大量在美国流通的芬太尼药丸以及制做芬太尼的原料和压药丸机等设备，居然都来
自中国。
据美国疾病预防管制中心（CDC）统计，美国因吸食过量芬太尼致死的人数在2013-2014
年间暴增了80%，相较之下，死于吸食过量海洛因的人数只成长了26%。
根据《华尔街日报》报导，芬太尼（fentanyl）的毒性是海洛因的50倍，去年春天中国
海关查获了70公斤芬太尼和乙醯芬太尼（acetyl fentanyl），由于没有采取适当防护
措施， 6名海关人员事后发病，其中1人陷入昏迷。美国缉毒署表示，该批芬太尼藏在
货柜内，走私的目的地是墨西哥，出货者为了躲避检查，透过5家物流商辗转送到海关
。经过调查，研判该批芬太尼的背后有庞大的毒品分销网络，源头都来自中国，走私到
墨西哥之后，再辗转进入美国。 |
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发帖数: 1 | 49 可以促进性功能
Erectile Dysfunction
Preliminary studies suggest propionyl-L-carnitine may help improve male
sexual function. One study found that carnitine improved the effectiveness
of sidenafil (Viagra) in men with diabetes who had not previously responded
to Viagra. In another study, a combination of propionyl-L-carnitine and
acetyl-L-carnitine improved the effectiveness of Viagra in men who had
erectile dysfunction after prostate surgery. More studies are needed. |
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