b*****l
发帖数: 9499 | 1 赞 boos :)
建议先用 DAPI image 来做 thresholding 找出每个 nuclei，可能还需要做
segmentation 来区分开相邻的 nuclei，这个用 watershed 比较好。然后用 nuclei
的结果做 mask 来处理 TUNEL 的图像（如果背景干扰比较大，而且背景跟信号有叠加
，可以先去一遍背景），就可以得出每个 nucleus 里的 TUNEL 信号来。把这 1000+
nuclei 的 TUNEL 信号统计起来画成一个分布，然后就可以用分峰软件准确地找到两个
峰，算出面积，就可以很准确地得出 apoptosis rate。同时，也可以找到最佳的
threshold 点来分 TUNEL 信号，这样就可以把原始图上的每个细胞核都标为 pos 或者
neg。这样做的好处是根据分峰结果可以算出两类错误分别是多少。
这个流程的每一步，都是很规范很常用的工具，可以 google 到 ImageJ 的代码。不知
道有没有人集成过，反正我到最后还是懒得集成了。
staining
see |
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h****n
发帖数: 2552 | 2 A Study on Amitosis of the Nucleus of the Mammalian Cell
哪位大侠能拿到PDF格式的paper，请mail 小弟.*****[email protected] |
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w***e
发帖数: 269 | 3 从seattle那个ISB抄来的.呵呵
Systems biology is the study of an organism, viewed as an integrated and
interacting network of genes, proteins and biochemical reactions which give
rise to life. Instead of analyzing individual components or aspects of the
organism, such as sugar metabolism or a cell nucleus, systems biologists
focus on all the components and the interactions among them, all as part of
one system. These interactions are ultimately responsible for an organism&
acute;s form and functions. For |
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a***e
发帖数: 1010 | 4 In S. Pombe and A. Thaliana, miRNAs are able to induce histone modification
and heterochromatin formation, which eventually inhibit transcription.
However, it is still controversial whether miRNAs translocate into the
nucleus and inhibit trasncription in metazoans.
It is big leap if you can demonstrate this. |
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l*****a
发帖数: 1431 | 5 We have patient lymphblast cell line and want to see if some mRNA are stuck
in the nucleus. our plan is to In situ hybridyzation using this cell line.
But the problem is how to fix the cells on slides?
Any suggestion? Thanks a lot |
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m*******r
发帖数: 7495 | 6 科学也可以像精灵的舞蹈一样优美，一切取决于发现者的眼睛。
展开想象的翅膀，你可以为艺术在life science中寻找到落脚点。
一期science杂志的封面，展现出成千上万yeast重叠生长所诞生的奇观：造型别致，宛
如一朵纯洁无瑕的白牡丹在静静地绽放。
大自然，绿，应和着阳光雨露，清新脱俗，美丽动人，处处散发着流动的生命色光彩。
你是否想过，cell像什么？答案可以千奇百怪，有一种回答如此：
Cell像一个city。Town hall是nucleus，城内千军万马的总指挥台；transportation
system是细胞的cytoskeleton，协调主管能源物资运输；mail是messenger，肩负信息
流通的重任；警察局是cell的defense system，维护和平安定的生活；人就像protein
，在不同岗位发挥着各自的功能。
想象是在宏观与微观，现实与虚幻，过去与未来中跳跃奔放的五线谱。是引导life
science不懈发展的神女火炬之光。
人类一思考，上帝就发笑。但是，人若停止想象，上帝将不复存在。
PS: 题目取得太大了，现在写它水平还太浅。就此搁笔，也许两年后... 阅读全帖 |
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a**v
发帖数: 406 | 7 It's OK to use for detecting cytoplasmic protein. It's not good for nuclear
protein since passive lysis buffer does not lyze nucleus. |
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m*p
发帖数: 226 | 8 Thanks!
Anybody knows a tag that can drive the protein to the nucleus?
Has anybody use H2b(his-58) ?
先谢了！ |
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m*p
发帖数: 226 | 9 那是当然。 Histone 2B has to be expressed in nucleus and binds to DNA.
Thanks though! |
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s******y
发帖数: 220 | 10 做subcellular fractionation 提取nucleus， 我的target 蛋白应该在cytosol和
nuclear fraction 都有。
可是用kit，只看到核有，用自己找的一个protocol只看到cytosol 有，也不知道核蛋
白提出来没。
请问nuclear loading control用什么好呢？ 我借了个lamin a的抗体，居然只在
cytosol里面有，不合理啊。
作为nuclear loading control， anti－histone是不好点？ 谢谢了。 |
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V***b
发帖数: 3419 | 11 做nucleus/cytoplasm fractionation。我现在用lamin A/C做nuclear marker。有些人
用lamin B或者histone。用什么最好呢？细胞质我用tubulin，有人用b-actin，但我脑
子里的印象actin在胞质/胞核都有吧？我现在做几个转录因子，在胞质/胞核之间跑来
跑去的，想找比较理想客观的marker。有经验的同学给说说。 |
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A******y
发帖数: 2041 | 12 There is actin in the nucleus? I did a lot cyto and nuclear extracts and using sigma actin ab (the strongest one out there) you can barely see a band while the cyto is like 1 sec exposure...Submit your paper with actin as nuclear loading control, let's see what the
review will say. |
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D*a
发帖数: 6830 | 13 现在很多根据结构的atlas，什么这个nucleus那个结构的，有没有功能结构图啊？
稍微详细点儿的，比如 xxx positive neuron是在什么什么地方，这类的，而不是大的结构。
具体问题是我想看看hypothalamus的在哪里的什么neurons是干嘛的，查文献查烦了。
。。 |
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w**t
发帖数: 52 | 14 只定位于Nucleus的蛋白也是在cyto合成后转运进核的吧。应该有可能吧，至少从空间
上说不矛盾。还可能间接调控呢。 |
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c********b
发帖数: 363 | 15 I feel it is not easy to conclude with current knowledge.
I knew two reference:
1, Mature blood cell has a capacity for splicing, even without nucleus.
This is on a 2005 Science paper
2, Last year, people showed mature blood cell has some sort of circadian
rhythm, suggesting that circadian is not maintained at transcription level,
but post transcriptional level. Last year one Science paper.
So our current knowledge may not be enough to explain. |
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a********k
发帖数: 2273 | 16 花了一个小时，深深的鄙视一下自己的无聊行径！！
125 蔡亮 男 1980年11月 复旦大学 生命
科学 2007年12月毕业于[美国]北卡大学 [美国]加州大学旧金山分校 博士后
Cai L, Mostov K. Polarity is destiny. Cell. 2009 Nov 13;139(4):660-2. PubMed
PMID: 19914162; PubMed Central PMCID: PMC2900917.
Cai L, Makhov AM, Schafer DA, Bear JE. Coronin 1B antagonizes cortactin and
remodels Arp2/3-containing actin branches in lamellipodia. Cell. 2008 Sep
5;134(5):828-42. PubMed PMID: 18775315; PubMed Central PMCID: PMC2570342.
Cai L, Makhov AM, Bear JE. F-actin... 阅读全帖 |
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n***w
发帖数: 2405 | 17 I am not sure about nuclear factor.
I guess transcription factor and nuclear transcription factor are
interchangeable in most cases.
They can be expressed and latent in the cytoplasm and translocate to the
nucleus upon activation, for example, STAT3, NF-kB.
Also they can be nuclear encoded but transported to mitochondria to direct
transcription like Tfam. |
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n****n
发帖数: 434 | 18 some proteins (residing in the cytoplasm in non-mitotic cells) can be
wrapped into the nucleus at/after mitotic exit. |
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m******5
发帖数: 1383 | 19 I don't think this is always the case:
at least they should manifest some important function to be stabilized
inside cell nuclear.
A lot of other proteins can be wrapped into the nucleus but soon been
degraded |
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s******a
发帖数: 472 | 20 http://www.cell.com/molecular-cell/fulltext/S1097-2765(11)00331
Fifty Years after Jacob and Monod: What Are the Unanswered Questions in Molecular Biology?
=====================================================================
Tomorrow's Molecular Biology
Marc W. Kirschner
Harvard University
Today, the term molecular biology has a rather prosaic sound. After all,
there is little in contemporary biology that isn't molecular. It's hard to
remember that 50 years ago, molecular biology was distinctly ... 阅读全帖 |
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g*********5
发帖数: 2533 | 21 What are lentiviral vectors?
Lentiviral vectors are a type of retrovirus that can infect both
dividing and nondividing cells because their preintegration complex (virus
“shell”) can get through the intact membrane of the nucleus of the target
cell. Lentiviruses can be used to provide highly effective gene therapy as
lentiviruses can change the expression of their target cell's gene for up to
six months. They can be used for nondividing or terminally differentiated
cells such as neurons, ma... 阅读全帖 |
|
|
H******d
发帖数: 13 | 23 Thank you all for your suggestions!
Bingo! I succeeded in observing the GFP-localization of in nucleus.
The project seems in a good stage right now. Probably, this unknown function
protein is a DNA binding protein. Since completely unknown function and no
idea about the potential DNA target, I am thinking of that protein-DNA pull-
down assay might shed some sort of light. Actually I raised both functional
GFP and Myc tagged transgenic lines. If the tagged protein is labeled with
the affinity tag... 阅读全帖 |
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m*********n
发帖数: 158 | 24 哪个兄弟姐妹给个protocol把，主要想看一个protein 在不同得condition 下得
cytosol 和plamsic membrane enrichment,
有好得protocol或是好得kit可以推荐一下把，
谢谢 |
|
l**********1
发帖数: 5204 | 25 //webdoc.nyumc.org/nyumc/files/sun-lab/attachments/CPCB.ch03.Cell%
20Fractionation.pdf |
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l**********1
发帖数: 5204 | 26 HTTP://www.sendspace.com/file/k8dabu
that 10.9 MB pdf file now on there. |
|
|
c********r
发帖数: 189 | 28 1) FRAP to see the protein's mobility in nucleus.
2) cell fractionation to see if the protein only in DNA binding fraction |
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w******y
发帖数: 2504 | 29 As you suggested, maybe because of co-factors. Or, the A protein is
trunkated when it is moving into the nucleus. |
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w******y
发帖数: 2504 | 30 As you suggested, maybe because of co-factors. Or, the A protein is
trunkated when it is moving into the nucleus. |
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l**********1
发帖数: 5204 | 31 Bingo
人的血红细胞的生物钟韵律调控都可以不用DNA 的 transcription-translation feedback loops
各个血红细胞应该不会只有一套proteomics 不然生物钟韵律调控如何实现?
Nature. 2011 Jan 27;469(7331):498-
Circadian clocks in human red blood cells.
//www.ncbi.nlm.nih.gov/pubmed/21270888
full text link:
//www.dormivigilia.com/wp-content/uploads/2011/02/nature09702.pdf
Abstract
Circadian (～24 hour) clocks are fundamentally important for coordinated physiology in organisms as
diverse
as cyanobacteria and humans. All current models of the molecular circadi... 阅读全帖 |
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h***e
发帖数: 232 | 32 i'm a new to this.
Just wondering if DAPI can get into live mammalian cells and stain the
nucleus? If yes, why many protocols require cell fixation and
permeabilization?
Will DAPI do anything to membrane protein if it stains live cells?
Many thanks! |
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i**y
发帖数: 71 | 33 Interesting... There's also a lot of debate about miRNA functions in the
nucleus. |
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u**********d
发帖数: 573 | 34 nucleus可以用histone，lamin B
membrane没做过 |
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r****r
发帖数: 36 | 35 if you can stain it with DAPI, they must be DNA. under what circumstance you
can find DNA outside the nucleus? :) |
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j*******g
发帖数: 10 | 36 please email to j*****[email protected], thanks a lot!
Growth Factors. 2004 Sep;22(3):151-5.
Thrombopoietin signalling in physiology and disease.
Fishley B, Alexander WS.
Source
The Walter and Eliza Hall Institute for Medical Research, IG Royal Parade,
Parkville, VIC. 3050, Australia.
Abstract
Thrombopoietin (TPO) is a key physiological regulator of steady-state
megakaryocytopoiesis, the process of megakaryocyte production and maturation
that ultimately results in the formation of platelets. Ten years ha... 阅读全帖 |
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l**********0
发帖数: 135 | 37 转信息
姓 名 傅瑾
性 别 女
出生年月
专业技术
职 务 教授
所获学位 博士
授予单位 美国哥伦比亚大学
研究方向
学科专长
联系电话 0592-2187210
E - MAIL f***[email protected]
通信地址 厦门大学路168号文正楼502
邮 编 361005
个人中文简介
傅瑾，1967年生人，闽江学者。1999年10月，在美国哥伦比亚大学获博士学位。
1999—2000年，纽
约哥伦比亚大学神经与遗传发展学院，博士后；2000—2001年，美国加利福尼亚大学药
学系做博士后；
2001—2003年，美国加利福尼亚大学药学系副研究员；2002—2008，在美国 Kadmus 制
药公司任顾问；
2003-2008，美国加利福尼亚大学药学系任研究员；2005年10月29日，厦门大学医学院
任特聘讲座教
授。2009年9月1日，厦门大学医学院任特聘教授。在国际重要杂志上发表研究论文30篇
(SCI收录），包括Nature、Nature系列杂志等。主持科研项目经费（折合成人民币）
1000万... 阅读全帖 |
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b****s
发帖数: 148 | 38 是nucleoli，DAPI或Hoechst都染的
每个nucleus里边可以有很多个 |
|
l**********1
发帖数: 5204 | 39 Sounds reasonable.
But key point is 让人怀疑作者检测到的human HBV infected cccDNA是否是真的
human HBV infected cccDNA?
details please refer
. 文章中用Southern blotting检测到了cccDNA的形成。cccDNA是HBV转录的膜板，被认
为是HBV持续感染的关键因素。从结果来看cccDNA的形成时间与HBV蛋白、RNA和DNA相比
比较早，尤其量很高。其实对于HBV来说哪怕是CMV启动的HBV（比如pCMV-HBV）cccDNA
的量都是很低的，更不要说是自身启动子启动的了。在NTCP介导的HBV复制水平整体很
低的情况下，具有高水平的cccDNA形成，让人怀疑作者检测到的cccDNA是否是真的
cccDNA，所以作者必须证明他们所指示的条带是真的是cccDNA，必须用EcoRI酶切（
cccDNA能被EcoRI线性化）和加热煮沸cccDNA（沸腾后不会被解成单链）来确证，同时
必须要有阳性对照。当然对于鸭乙肝病毒（DHBV）
http://blog.scien... 阅读全帖 |
|
l**********1
发帖数: 5204 | 40 Sounds reasonable.
But key point is 让人怀疑作者检测到的human HBV infected cccDNA是否是真的
human HBV infected cccDNA?
details please refer
. 文章中用Southern blotting检测到了cccDNA的形成。cccDNA是HBV转录的膜板，被认
为是HBV持续感染的关键因素。从结果来看cccDNA的形成时间与HBV蛋白、RNA和DNA相比
比较早，尤其量很高。其实对于HBV来说哪怕是CMV启动的HBV（比如pCMV-HBV）cccDNA
的量都是很低的，更不要说是自身启动子启动的了。在NTCP介导的HBV复制水平整体很
低的情况下，具有高水平的cccDNA形成，让人怀疑作者检测到的cccDNA是否是真的
cccDNA，所以作者必须证明他们所指示的条带是真的是cccDNA，必须用EcoRI酶切（
cccDNA能被EcoRI线性化）和加热煮沸cccDNA（沸腾后不会被解成单链）来确证，同时
必须要有阳性对照。当然对于鸭乙肝病毒（DHBV）
http://blog.scien... 阅读全帖 |
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n********e
发帖数: 1630 | 41 HBV跟HIV不一样，HIV有intergrase，可以整合到host 基因组里，HBV 没有，cccDNA独
立存在于nucleus
确实，在美国读到的宣传，HBV和HIV的传播途径一样，但是HBV 感染能力比HIV强100倍
。没有说是共餐可以传播的。但是我记得小时候在国内的时候，大家都说共餐会传染，
也就是密切接触。但是我在美国从来没有听过这样的说法。那么中国有那么多乙肝患者
，是怎么回事呢？ 难道是不会用condom的原故？
|
|
p****s
发帖数: 3153 | 42 My advisor says peenas a lot. And for 恶俗,
look at the acronym here:
Proton-enhanced nuclear induction spectroscopy
Proton-enhanced nuclear induction spectroscopy (PENIS[1]) is a nuclear
magnetic resonance technique invented by Michael Gibby and Alexander Pines
while they were graduate students in the lab of Professor John S. Waugh at
the Massachusetts Institute of Technology.[2] It was one of the first of
Pines' experiments transferring spin orientation from one atomic nucleus to
another, which ... 阅读全帖 |
|
j***x
发帖数: 1469 | 43 Cell
Volume 71, Issue 3, 30 October 1992, Pages 527–542
Cover image
Article
The human XIST gene: Analysis of a 17 kb inactive X-specific RNA that
contains conserved repeats and is highly localized within the nucleus
Carolyn J. Brown★, †,
Brian D. Hendrich★, †,
Jim L. Rupert★, †,
Ronald G. Lafrenière★, †,
Yigong Xing‡,
Jeanne Lawrence‡,
Huntington F. Willard★, †
Please send to l************[email protected]
Thank you very much! |
|
l**********1
发帖数: 5204 | 44 BMJ
Gut is not trash journal
this journal 2013 one review
web link:
http://gut.bmj.com/content/62/8/1093.extract
its pp1094 left column top
cited here stuff sentence,
>Surprisingly, Yan et al did not provide
>Southern blot data demonstrating accumulation
of HBV DNA replication intermediates,
as this electrophoretic pattern is
a signature of productive virus replication.
Only one subfigure shows some evidence
of cccDNA formation, which
defines a successful infection by HBV;
however, unlike HBV DN... 阅读全帖 |
|
o**4
发帖数: 35028 | 45 一公超过1000的还真没有，似乎都是review：
这些事一公引用率前4的文章，似乎都是review：
Mechanisms of TGF-β signaling from cell membrane to the nucleus
Y Shi, J Massagué - Cell, 2003 - Elsevier
TGF-β signaling controls a plethora of cellular responses and figures
prominently in animal
development. Recent cellular, biochemical, and structural studies have
revealed significant
insight into the mechanisms of the activation of TGF-β receptors through
ligand binding, ...
Cited by 3259 Related articles All 25 versions Cite
The galvaniza... 阅读全帖 |
|
p*****c
发帖数: 20445 | 46 Christopher D. Lima, Ph.D.
Memorial Sloan-Kettering Cancer Center
Christopher Lima doesn’t pick easy problems. A structural biologist, he is
investigating how cells attach small proteins—like ubiquitin and the
related molecule SUMO—to other proteins to modify their function or fate.
Alongside those studies, he is exploring the molecular mechanisms that
underlie RNA processing and degradation—modifications to an RNA copy of a
gene that influence its stability and ability to be used as a template ... 阅读全帖 |
|
m******g
发帖数: 467 | 47 今天去找导师汇报，本来觉得自己的内容太少，会被批评的，结果导师还鼓励了我，我
一定要加倍努力呀！感动ing。希望以后也能成为我导师那样的好导师！
Science 13.12.13
推荐：
1. Exonic Transcription Factor Binding Directs Codon Choice and Affects
Protein Evolution
概要：Long-Term Dynamics of Adaptation in Asexual Populations
Mean fitness seems to have no upper bound
Power law better than hyperbolic model
2. The Hidden Codes That Shape Protein Evolution
TF binding code
Human genome in 81 cell types
Constraining codes
~14% codons within 86.9% of genes are occupied by TFs
3. Cave... 阅读全帖 |
|
m******g
发帖数: 467 | 48 今天去找导师汇报，本来觉得自己的内容太少，会被批评的，结果导师还鼓励了我，我
一定要加倍努力呀！感动ing。希望以后也能成为我导师那样的好导师！
Science 13.12.13
推荐：
1. Exonic Transcription Factor Binding Directs Codon Choice and Affects
Protein Evolution
概要：Long-Term Dynamics of Adaptation in Asexual Populations
Mean fitness seems to have no upper bound
Power law better than hyperbolic model
2. The Hidden Codes That Shape Protein Evolution
TF binding code
Human genome in 81 cell types
Constraining codes
~14% codons within 86.9% of genes are occupied by TFs
3. Cave... 阅读全帖 |
|
b******j
发帖数: 361 | 49 【 以下文字转载自 Immigration 讨论区 】
发信人: baggiosj (baggio), 信区: Immigration
标 题: 请帮忙下载几篇文章， 在线等
发信站: BBS 未名空间站 (Tue Jan 7 23:13:07 2014, 美东)
1. FGF, TGFβ and Wnt crosstalk: embryonic to in vitro cartilage development
from mesenchymal stem cells, journal of tissue engineering and regenerative
medicine.
DOI: 10.1002/term.1744
2. Enhancing human nucleus pulposus cells for biological treatment
approaches of degenerative intervertebral disc diseases: a systematic review
，journal of tissue engineering and r... 阅读全帖 |
|
l*******i
发帖数: 153 | 50 有意思。
是female mESC吗？是的话，先做两个实验：
1）H3K27me3 immunostaing：可能发现small dot in nucleus.
2)qRT-PCR测Xist的表达，或者用FISH。
证实的话，就说明出现X chormosome inactivation了。
几个问题：
3) mESC的培养条件？serum,还是2i/LIF?
1）knockdown的mESC是否仍保持未分化状态，还是开始分化了？如果未分化，那么很有
可能你发现了一个新的调节X reactivation/inactivation的因子，做深了，至少是
cell stem cell.如果细胞分化了，那么X chromosome上的genes表达下调不奇怪，有多
大新意取决于你的这个gene
2）看一下这个基因的功能，是否已有先关报道，与X chromosome reactivation有关。
是histone modifier, TF还是在X chromsome上的新lncRNA? |
|
