A******y
发帖数: 2041 | 1 Almost all the modern western blot imaging system is better than films (
assuming you are using femto level substrate), I like the top of the line
alpha-innotech imager (do not get the red or blue one they try to sell; they
are horrible in my opinion). Bio-rad imager is very common but I think
their tech is the oldest. GE one is very nice. Do not get Kodak one if it
is still on the market (horrible).
Bio-rad recently join the ECL reagent market, their femto-ECL (Clarity) is a
lot cheaper than... 阅读全帖 |
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b****u
发帖数: 2771 | 2 curious question: what is time scale for kinase and substrate binding. |
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S*********s
发帖数: 304 | 3 many kinase/substrate interaction is transient
can not get interaction by IP unless using crosslinker |
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a******a
发帖数: 283 | 4 any trick to reduce the noise generated by the genomic DNA background? There
is no gene of interest in the genome, the primers only anneal to the viral
gene in the plasmid. I added the genomic DNA as background to generate
standard curve for future study (when measure genes instered in the genome).
However there is a lot of background when added the genomic DNA.
Primers work fine with only plasmids as DNA substrate. |
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A******y
发帖数: 2041 | 5 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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A******y
发帖数: 2041 | 6 But there are a lot papers literally are looking at SIRT5 and SIRT6 as
deacetylases (they are
not!) Just becasue you artificially increase the substrate concentration in
a test tube and get some activity, does not mean they are deacetylase. Btw
, there are Cell papers look at SIRT5 as deacetylase in-vivo, and in my
opinion, they are looking at other SIRT or HDAC that binds SIRT5. SIRT and
HDACs are all in a huge complexes.
Btw, the same thing for HDAC class IIa, HDAC8, HDAC6 domain 1, HDAC10,... 阅读全帖 |
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A******y
发帖数: 2041 | 7 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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b******y
发帖数: 627 | 8 What emases meant is that A or B or both might allosterically regulate ATP'
s binding affinity to kinase and therefore the Km of ATP can be different
when A or B as substrate due to differential allosteric effects from A and B
. |
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A******y
发帖数: 2041 | 9 Because both acetate and succinate are also mitochondrial substrates. For
example, I think succinate protects kidney cells from oxidative injury ex
vivo. Acetate could do they same thing. Yes, both of them will get
converted to tocopherol. Btw, tocopherol is the bad vitamin E. It is the
one has been shown to increase your prostate cancer rate. If you really
want to protect cells, try tocotrienol. You will realize it is at least 10
to 100 times stronger. The drug Edison pharmaceutical tryi... 阅读全帖 |
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f******g
发帖数: 382 | 10 Yes.
X -- substrate concentrations (cover at least 1/2Km to 5Km)
Y-- initial velocity
fit to Michaelis–Menten kinetics, Km will be determined. |
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l****y
发帖数: 398 | 11 thanks a lot!
Expert Rev Proteomics. 2008 Jun;5(3):497-505. doi: 10.1586/14789450.5.3.497.
Identification of protein kinase substrates by proteomic approaches. |
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C*******e
发帖数: 4348 | 12 用AP conjugated 2nd 哪有那么烂
是一抗太差吧
HRP的如果信号太强的带还可能很快quench呢
另外lz:
用HRP的也有substrate可以买来显色做colometric的 |
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h*****c
发帖数: 505 | 13 要多少钱啊？
都要买些什么东西啊
我们这边做一些表面处理的试验，想检测一下处理后材料的生物相容性什么的，准备做
一下cell culture study, 下面是从文献中找到的试验方法
Cell culture experiments were performed using mouse pre-osteoblast
MC3T3-E1 subclone 14 and rat fibroblast Rat1 cell lines provided by
American Type Culture Collection (ATCC, Manassas, VA, USA). Disks
of the titanium substrates with a diameter of one centimeter were placed in
24-well culture plates and incubated in alpha minimum essential medium
(a-MEM, Invitrogen Corporation, USA) and Dulbecco’s m... 阅读全帖 |
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p*****h
发帖数: 36 | 14 Raf has very few known substrates (MEK)
For the majority, specificity is determined by the activation segment of the
kinase domain. |
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p*****e
发帖数: 93 | 15 more than MEK's substrate.
the |
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p*****h
发帖数: 36 | 16 You mean not just MEK?
Raf does not have a specificity motif. Peptide library approaches give no
definitive results. - this is generally taken as evidence that sequences
outside the kinase domain help anchor substrates. |
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p*****h
发帖数: 36 | 17 2-DG is a substrate of HK, not an inhibitor. But it cannot be further
metabolized by downstream enzymes. In this sense, it inhibits glycolysis.
DCA is dirty. So is TZD.
glycolysis |
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p*****h
发帖数: 36 | 18 Suicide inhibitor is a term reserved for those capable of covalent
modification of enzyme active site. Of course at high concentrations, 2DG6P
as a substrate /product analogue inhibits several glycolytic enzymes
including HK and PFK.
It is for historical reasons that these drugs are dirty (phenotypic drug
screening came well before rational design). This is not to say there will
be no better drugs.
在 hellozero (hellozero) 的大作中提到: 】
s
inhibitors/ |
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T****u
发帖数: 424 | 19 很多死法，做Annexin V and PI staining
另外看有没有caspase activation with western of cleaved Caspase or luc
substrate.
不是所有的death都值得研究的，除非，你看到的death有具体的pathway
我们做screen的知道，很多compound扔下去细胞都会死。 |
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d********n
发帖数: 15 | 20 it's not degradation (proteases work less efficiently at ~90C, but will be a
disaster at ~55C at which proteases have reduced activity but protein
substrates begin to open structure and give much higher accessibility), but
precipitation because proteins lose native structure under heating. This is
why SDS or LDS loading bf needs to be added before heating. 1g sds binds to
14g proteins, 10ul sds loading bf is supposed to be enough wrap all proteins
in
the sample as they are so low.
the only probl... 阅读全帖 |
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S******i
发帖数: 71 | 21 1
Lysine Glutarylation Is a Protein Posttranslational Modification Regulated
by SIRT5.
Cell Metab. 2014 Apr 1;19(4):605-17. doi: 10.1016/j.cmet.2014.03.014.
2.
Lysine 2-hydroxyisobutyrylation is a widely distributed active histone mark.
Nat Chem Biol. 2014 May;10(5):365-70. doi: 10.1038/nchembio.1497.
3
Identification of 67 histone marks and histone lysine crotonylation as a new
type of histone modification.
Cell. 2011 Sep 16;146(6):1016-28. doi: 10.1016/j.cell.2011.08.008.
4
The first identific... 阅读全帖 |
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r*********g
发帖数: 1160 | 22 请教一下大家一下，有两个功能是需要的：
1. 根据已有晶体结构的homologous protein预测目标蛋白结构？
2. 在蛋白上做突变，然后看其与新substrate的结合，需要重新模拟结构，比较和WT的
变化。
我以前用过MOE，Insight 2， 但大概是快8年前了，想问一下现在大家用什么软件，有
免费的更好，非常感谢。 |
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g*********5
发帖数: 2533 | 23 Isolation of nuclei for the co-IP and whole ChIP protocols is based on the
methods of Gendrel et al. (2002, 2005), Johnson et al. (2002), and Nelson et
al. (2006).
METHOD
For extraction and co-IP of nuclear proteins, see Steps 1-39 (Fig. 1). For
the ChIP procedure, see Steps 40-69 (Fig. 2).
Figure 1.
View larger version:
In this page
In a new window
Figure 1.
Flowchart for the timeline and organization for co-IP of nuclear proteins
from Arabidopsis seedlings.
Figure 2.
View larger versio... 阅读全帖 |
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q******g
发帖数: 3858 | 24 解结构，有可能发现substrate binding pocket(site),然后根据这个结合位点地结构
用软件来筛选特异性地抑制剂，然后再通过实验来验证。这样，筛选过程能省不少时间。
而且，她做的人源GLUT1还是很难做的。我一个朋友做另一个膜蛋白，做了3年多了，还
没有拿到晶体呢。 |
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l****5
发帖数: 2968 | 25 下面很多papers，没有学校账户查不到了。请大家帮忙查一下吧。请发greenfast2013@
gmail.com
包子不多，不过由于文章很多，所以会发包子以表感谢的。万分感谢啊！
1) Effects of high electric field pulses on enzymes
2) Neuropharmacological investigations on muscimol, a psychotropic drug
extracted from Amanita muscaria
3) Extraction of BaChu mushroom polysaccharides and preparation of a
compound beverage
4) The effect of the high pressure homogenisation on the activity and
stability of a commercial neutral protease from Bacillus subtilis
5) Isolation o... 阅读全帖 |
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m******g
发帖数: 467 | 27 Nature 14.09.18
稍微了解一下：
1. Genome sequencing of normal cells reveals developmental lineages and
mutational processes
2. Proteogenomic characterization of human colon and rectal cancer
小知识：
1. The genomic substrate for adaptive radiation in African cichlid fish
2. When disease strikes from nowhere |
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m******g
发帖数: 467 | 28 Nature 14.09.18
稍微了解一下：
1. Genome sequencing of normal cells reveals developmental lineages and
mutational processes
2. Proteogenomic characterization of human colon and rectal cancer
小知识：
1. The genomic substrate for adaptive radiation in African cichlid fish
2. When disease strikes from nowhere |
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s********3
发帖数: 231 | 29 《科学》对STAP手稿的专家评审意见曝光
《科学》对传说中的STAP细胞论文的三名评审专家意见，过去一直被雪藏，今天终于
被曝光。专家对这一研究的评价不高，认为非常全面的描述性研究，如果结果确定，可
能导致发育生物学大厦的颠覆，其实是不相信这种研究结论。因为没有当时的投稿件，
所以没有办法对这些评价进行更深入分析。不过这些资料显然都在某个地方，只是拥有
的人不愿意拿出来。
Retraction Watch readers are of course familiarwith the STAP stem cell saga,
which was punctuated by tragedy last month whenone of the authors of the
two now-retracted papers in Nature committed suicide.
In June, Science‘s news section reported:
Sources in the scientific community confirm that early version... 阅读全帖 |
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w********h
发帖数: 12367 | 30 stress-induced stem cell differentiation很多了，
我也有文章和这方面相关，substrate stiffness的作用。
正在看Scientific American上面的一篇文章Twists of Fate，
里面讲了一些我原本不知道的东西，比如YAP/TAZ这个duo protein在其中的作用。
这篇文章也提到了自杀了的Yoshiki Sasai的工作。 |
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w********h
发帖数: 12367 | 31 stress-induced stem cell differentiation很多了，
我也有文章和这方面相关，substrate stiffness的作用。
正在看Scientific American上面的一篇文章Twists of Fate，
里面讲了一些我原本不知道的东西，比如YAP/TAZ这个duo protein在其中的作用。
这篇文章也提到了自杀了的Yoshiki Sasai的工作。 |
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r******k
发帖数: 446 | 32 估计pi3k pathway 也没我想想的那么简单吧。。 有可能下游的很多substrate也已经
被激活了 在糖尿病人里面？
关。 |
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g*****n
发帖数: 241 | 33 http://www.cell.com/cell/pdf/S0092-8674(14)01367-1.pdf
A Variety of Dicer Substrates in Human and C. elegans
please email to [email protected]
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/* ]]> */
thank... 阅读全帖 |
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A******y
发帖数: 2041 | 34 I won't cite a site that spells drug name wrong. If you look into those FDA
approved drugs, they are all have a lead molecule (already known or exist)
to work with or already exist in a HTS library (Gleevec). If you found a
new target and there is absolute no lead, you are in trouble. Btw, sometime
understanding the biology of the target will help because the substrate of
the target usually will give you some lead. |
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s********9
发帖数: 132 | 35 When people say there are hundreds E3 in the proteome, do they know each and
every one in these hundreds has proven activity
towards some sort of substrate? Otherwise where does this number (~ one
thousand or some say ~600) come from?
Also,
For SCF, people say there are hundreds of them, do they know experimentally
the S, C, and F of each SCF? otherwise where does
this number (few hundres SCF) come from? (Is it the theoretical combinations
of all S,C,F?)
The same question of CRL. Do they know th... 阅读全帖 |
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S*******r
发帖数: 530 | 36 1. 有RING domain的被假定为E3，通过genome wide search
2. 能autoubiquitylation的被认为是E3
3. 很多E3还没有发现substrate |
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z*****g
发帖数: 2 | 37 Allosteric modulators具有高选择性、低毒性等特有优势，最近几年已经成为药物研
发的热点领域。但目前主要以偶然发现为主，还很难对一个蛋白的potential
allosteric pocket （different from the protein's active site）进行定向的drug
design。我们组构建了一个综合性的AlloSteric Database http://mdl.shsmu.edu.cn/ASD/生物信息学平台，包括allosteric targets, modulators, binding site, Mechanism, Disease and drugs；并在陆续开发相关的计算服务工具，例如，预测蛋白的allosteric sitehttp://202.120.138.35/AST/，用于Virtual Screening，定向筛选Allosteric modulator。
其实，我们最近设计获得的一个小分子activator for epigenetic enzyme，最开
始就是我自己predict 这个酶的... 阅读全帖 |
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w***r
发帖数: 709 | 38 这个人太牛X了。他在song h 甲基化文章真的很炫!！
但是我个人觉得
Cytochrome P450 drives a HIF-regulated behavioral response to reoxygenation
by C. elegans. Science (2013) Vol. 341 no. 6145 pp. 554-558. PMID: 23811225
这篇文章似乎新意不够，hif 和p450和缺氧的关系早就知道了。下面是一篇，还有不少
有明白人讲一讲吗？
Inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-stimulated Cyp1a1
promoter activity by hypoxic agents.
Kim JE1, Sheen YY.
Author information
Abstract
Since hypoxia-inducible factor-1alpha (HIF-1alpha) and the arylhydrocarbon
receptor (AhR) shared th... 阅读全帖 |
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w***r
发帖数: 709 | 39 这个人太牛X了。他在song h 甲基化文章真的很炫!！
但是我个人觉得
Cytochrome P450 drives a HIF-regulated behavioral response to reoxygenation
by C. elegans. Science (2013) Vol. 341 no. 6145 pp. 554-558. PMID: 23811225
这篇文章似乎新意不够，hif 和p450和缺氧的关系早就知道了。下面是一篇，还有不少
有明白人讲一讲吗？
Inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-stimulated Cyp1a1
promoter activity by hypoxic agents.
Kim JE1, Sheen YY.
Author information
Abstract
Since hypoxia-inducible factor-1alpha (HIF-1alpha) and the arylhydrocarbon
receptor (AhR) shared th... 阅读全帖 |
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l**n
发帖数: 109 | 40 我觉得和pH还有ionic strength有关。enzyme, inhibitors, and substrate in
identical solvent? |
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w********h
发帖数: 12367 | 41 谢谢科普。我们做生物材料的，觉得生物物理的人做的已经很明白了呢。
怎么解释neuronal cells更喜欢softer substrates?
focal adhesion不是universial?
rigidity
nm |
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s***e
发帖数: 911 | 42 我也是外行。正在学. Focal Adhesion肯定不universial. 在2D表面上，需要
substrate够硬度才行。还需要frineticin或者collagen coating才行。有时候在没有
coating的表面上实验的粘连，其实是因为细胞局部modify了表面后产生的。 |
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X******n
发帖数: 914 | 43 Lack of Evidence for PKM2 Protein Kinase Activity－Molecular Cell
Highlights
•PEP-dependent phosphorylation was assayed in cell lysates using
radioactive PEP
•Trace adenine nucleotides can confound PEP-dependent phosphorylation
assay results
•PEP-dependent phosphorylation is rare and is not dependent on PKM2
•We are unable to detect PKM2 protein kinase activity in a variety of
contexts
Summary
The role of pyruvate kinase M2 (PKM2) in cell proliferation is controversial
. A... 阅读全帖 |
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z********i
发帖数: 610 | 44 我只能说你们是毫无根据的猜想，里事实相差的很远。有人说他有一个博后想回去，被
committee否了，实际上人家根本就不可能回去，去面试也是抱着打酱油的心态。
liang的Figure2F 2H不会有问题，很容易就能重复出来，3G这个就不好说，就好比
kinase and substrate，transit interaction本来就不容易检测。
最后的结局可能是梁想自爆，但是没有成功，对冯有一定的影响。 |
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l*****7
发帖数: 8463 | 45 1
http://disinfo.com/2011/05/could-human-photosynthesis-power-the-future/
Could Human Photosynthesis Power The Future?
Posted By: phunkychic666 MAY 4, 2011
Chlorophyll. Photo: Wilfredo R. Rodriguez H. (CC)
Chlorophyll. Photo: Wilfredo R. Rodriguez H. (CC)
By Hank Mills for Pure Energy System News:
Dr. Arturo Solis Herrera has developed a technology that utilizes the
properties of melanin – the animal analogue to chlorophyll. His melanin
based (polihydroxyindol) photo-cell absorbs a broad spectru... 阅读全帖 |
|
发帖数: 1 | 46 Try Ca for DNA experiments.
Try the same experiments shown on S figure 2 by using DNA instead of RNA as
a substrate.
Han Lab's Mg reagent may be contaminated with Ca or other metal cations.
Good luck to all |
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V******1
发帖数: 8 | 47 作为一个没做过膜蛋白的伪structure biologist，赞一下专业回答。
再补充一点生物大分子的。结构可能对于大分子比如抗体的指导作用没有receptor/
ligand或者enzyme/substrate那么看起来那么直接，但也有很大帮助。比如知道target
的结构，就可以有针对性地display更感兴趣的epitope，尤其是那些non-linear的
epitope单单根据序列是没法预测的。反过来说也可以突变掉unwanted epitope来避免
screen到不想要的抗体。拿到antibody/antigen complex的结构，可以根据结构做有针
对性的affinity maturation来进一步优化抗体。design bi-specific甚至tri-
specific antibody，需要考虑几个target epitope在空间上的位置。做vaccine的，可
以根据结构来design engineer的antigen，使之更有效地产生neutralizing antibody
。还有找到一个好的抗体，patent的时候也得知道epitope的不是，虽... 阅读全帖 |
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r**a
发帖数: 121 | 48 哪位同仁可以帮我下一下这个文章啊，麻烦发送到：
[email protected]
谢谢啦：）
FASEB J. 2017 Sep;31(9):4153-4167. doi: 10.1096/fj.201700063R. Epub 2017 Jun
7.
Sestrin2 prevents age-related intolerance to ischemia and reperfusion injury
by modulating substrate metabolism. |
|
a**********3
发帖数: 86 | 49 Mechanosensitivity of Cancer Cells in Contact with Soft Substrates Using AFM
Yara Abidine, Andrei Constantinescu, Valérie M. Laurent, Vinoth Sundar
Rajan, Richard Michel, Valentin Laplaud, Alain Duperray, Claude Verdier
DOI: https://doi.org/10.1016/j.bpj.2018.01.005
Biophysical Journal
Thanks! |
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p***z
发帖数: 94 | 50 急需
physica status solidi (c)
Volume 5 Issue 12, Pages 3708 - 3711
Title: Electronic structure investigation of nickel phthalocyanine thin film
interfaces with inorganic and organic substrates
请发我邮箱:a********[email protected]
非常感谢!! |
|
