j****1
发帖数: 15497 | 1 哈哈哈哈~
microtubules, actin filaments and microfilament are components of
cytoskeleton.
We don't say that it's in the cytoplasm. |
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r**r
发帖数: 171 | 2 A good CSF extract should be arrested @ metaphase.
After the addition of calcium or fertilization which causes a transient
increase in cytoplasmic calcium conc., the CSF will be inactivated, following
by APC activation, cyclin B destruction and mitotic exit. And this process is
roughly 30 min. It is easy to collect phosphorylated separase before the
addition of calcium. To collect the unphosphorylated one, like you say, you
can throw sperm chromatin into extract and monitor the progresstion by D |
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m*******s
发帖数: 6 | 3 In C.elegans, all kinds of expression vectors are available. N-terminalor
C-terminal fused, cytoplasm located or nuclear located. Usually, we use the
same 3'UTR. Sometime we will use the gene specific 3'UTR in order to know the
accurate expression pattern of that gene.
that |
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l********u
发帖数: 82 | 4 请问，高手能否拿到如下全文？不胜感激？
1。
Title: Pharmacological Enhancement of Cardiac Gap Junction Coupling Prevents
Arrhythmias in Canine LQT2 Model.
Author(s): Quan, XQ, Bai R, Lu LG et al
Source: Cell Communication and Adhesion. 2009;16(1-3):29-38.
2。
Title: Identification of Binding Partners for the Cytoplasmic Loop of
Connexin43: A Novel Interaction with beta-Tubulin.
Author(s): Kang, EY; Ponzio, M; Gupta, PP et al
Source: Cell Communication and Adhesion. 2008;15(5-6):397-406
3。
Title: Self-Assembly of Si |
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a***e
发帖数: 1010 | 5 (1) if your protein localizes in cytoplasm, a gentle lysis will only release
your protein to the solution, but not chromatin DNA.
(2) if your protein accumulates in nucleus but not sticks to DNA, you can
try several rounds freeze-thaw in some buffer with detergent. Then spin down
. The chromatin DNA will stay in pellet.
(3) and you can always add a pre-clear step with beads only. |
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a***e
发帖数: 1010 | 6 no method is perfect clean.
if you don't care about recovery efficiency, u can reduce the time of first
NP-40 treatment to get clean cytoplasm, but increase the time of second NP-
40 treatment to get clean nuclei. |
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a***e
发帖数: 1010 | 7 separate nuclei and cytoplasm by 0.5% NP-40/PBS for 5 min on ice. then
standard purification of RNA.
there are also kits for these, just google. |
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g********i
发帖数: 207 | 8 histone H3 is good. histone H1 is not good coz sometimes it is also located
in cytoplasm. |
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s******y
发帖数: 28562 | 9 Thanks!
The reason people are interested in RGD peptide is because it mimics the
extracellular matrix and binds to the integrins, I believe.
I actually don't really care what kind of peptide it is. However I am kind
of planning to do some pilot experiments by myself and I prefer to use
something that can be handled easily. Therefore, I would like to have
something that is sovlable and cytoplasmic (which will be easy for
expression and require minimal posttranslational modification). While the
ex |
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S*****s
发帖数: 287 | 10 I think it is project dependent, and probably investigator dependent, so it
is my personal opinion here. In mammalian cells, GFP shows up as soon as it
is translated, so it can be hard to tell membrane expression from
cytoplasmic expression. In my study, the wild type protein had efficient
membrane trafficking, and mutant protein decreased trafficking efficiency.
GFP fusion protein was not able to show the weak membrane expression of the
mutant protein. When I used HA-tagged protein, I detected ... 阅读全帖 |
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n********k
发帖数: 2818 | 11 What an AMAZING woman! seriously!! I vote her to be a next Nobel laureate
and a first Chinese and it is a girl too....
http://english.cas.cn/accessory/twows4th/twowsaward/201006/t20100627_55793.html
Professor Fanyi Zeng was born in 1968. She received her B.A. in University
of California San Diego, CA, USA in 1991. In 2005, she got her Ph.D in
University of Pennsylvania, PA, USA.
Professor Zeng has led a research team that has made substantial and long-
lasting contributions to the developmental ... 阅读全帖 |
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l*****i
发帖数: 1163 | 12 A transcription factor with SH2 and SH3 domains is directly activated by an
interferon alpha-induced
cytoplasmic protein tyrosine kinase(s).
Fu XY.
Cell. 1992 Jul 24;70(2):323-35.
这个悲剧英雄！ |
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b*********2
发帖数: 35 | 13 如标题。在哺乳细胞中。
能推荐一下参考文献吗？ |
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m******5
发帖数: 1383 | 14 恩，一般是用tublin作cytoplasmic marker
从方法上说这样没问题么？另外，核能裂完全么？ |
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s****z
发帖数: 50 | 15 Is histone mRNA equally distributed in nuclei and cytoplasm? |
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c*******s
发帖数: 29 | 16 I think you should use the same proportion of the nuclear RNA and
cytoplasmic RNA for the RT-PCR. What's the purpose of your control? You can
do RT-PCR for an pre-mRNA (containing introns which should be exported) to
show your fractionation is clean. |
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c*******e
发帖数: 4642 | 17 不是吧。有些有single sequence的，直接在cytoplasm合成以后就自己插进去了
ER |
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a*****t
发帖数: 2 | 18 我对实验不是很懂，没做过实验。
RNA测序所指的total RNA是不是包括细胞内的所有RNA？不区分Nuclear or
Cytoplasmic？如果不区分，那是不是核内的初生RNA也被测到了？ |
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X***n
发帖数: 366 | 19 Yes, it's true. Unless you do cytoplasm and nuclear extraction
separately. |
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H****s
发帖数: 301 | 20 现在在尝试用原核系统（E.coli)表达一个病毒蛋白（400aa)，T7 promoter，BL21(DE3
) host。现在遇到了一个难题，这个基因怎么也克隆不到原核表达载体里去。转化后有
colonies，但是小提质粒后发现没有插入片段。
首先排除的是克隆问题，同样的片段，同样的酶切位点，使用完全同样的试剂，该基因
很容易就克隆到真核表达载体里去。
尝试了cytoplasm表达载体，periplasm表达载体,和不同的宿主菌，都是同样的结果。
（原核表达老手了，头一次遇到这种情况）。
是不是此蛋白毒性太大，即使是一点点的leaky expression都不行？
我现在在想的试验是：（1）做一个这个基因的文库，来看看到底是哪部分有毒性；（2
）使用GroES的leader peptide试试看。
各位有没有什么建议？谢谢。 |
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a**v
发帖数: 406 | 21 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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y**********o
发帖数: 37 | 22 最近老板忽然对Alzheimer's disease产生极大的兴趣,非要把课题往这个方向上靠.我
研究了几篇综述后,有些最基本的问题想请教一下版上做这个方向的高人.相信这里应该
有很多前辈能够指导一下我这个外行,晚辈先在这里感谢一下所有回帖的.
问题一,目前市面上各种mouse models似乎都是转了human mutated genes,像APP,PS1什
么的.最常见的检测指标是A-beta和NFT的染色. 老鼠也有内源性的APP和Tau, 好象内源
性的Tau所形成的NFT在小鼠中一直无法诱导,那么内源性的APP可以经诱导产生A-beta斑
块吗? 公司能买到针对rodent的A-beta抗体, 说明有这种可能,只是主流的文章似乎都
在关注human gene product.有没有可能用老鼠内源性的APP来做疾病模型?
问题二,目前主流的观点对这个疾病的机理是否偏向于Ca overload? 我们手头现有的
mouse model显示reduced cytoplasmic Ca concentration,类似PSI/PS2 KO 模型,但这
个似乎无法被牵强的说成Alzhe... 阅读全帖 |
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w*****n
发帖数: 107 | 23 you can use RNA as a control, such as 47S rRNA or pre-mRNAs.
It is not control for loading, but control for the purity of fractionation.
If your cytoplasmic fraction is not contaminated by nuclear fraction, most,
if not all, of these RNAs should be in the nuclear fraction. |
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n***w
发帖数: 2405 | 24 would you please be more specific?
If it's inactive in the cytosol, and it is redistributed to membrane after
phosphorylation. It may be due to changes to cytoskeleton and motor proteins.
If it is normally a nuclear protein or cytoplasm protein, somehow it is
translocated into the subcellular compartment like mitochondria or other
organelles, it may interact with some proteins that have those translocation
signals. |
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m*p
发帖数: 226 | 25 线粒体的tRNA synthesis is different from cytoplasm tRNA synthesis? 谁有这方
面的背景， 来扫盲。 谢谢！ |
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V***b
发帖数: 3419 | 26 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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p*********n
发帖数: 556 | 30 用C18 ion pair, rentention time很不一样的说。 |
|
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p*********n
发帖数: 556 | 32 什么意思？
如果你extract all adenine nucleotides, 应该能看到ATP，ADP, AMP，GDP, GTP,
GMP, IMP...跑个standard mixture就知道它们的retention time啦。 |
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m******5
发帖数: 1383 | 33 google几篇做beta-catenin NES fusion protein的文章，用那个NES序列大概能行
不过好奇楼主是怎么判断出NES 序列不行的？
单个GFP是自由穿梭的，从cytoplasmic-nuclear distruibution未必能很明显地看出区别 |
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t****y
发帖数: 168 | 34 Check the paper:
Cytoplasm-localized SIRT1 enhances apoptosis
Journal of Cellular Physiology
Volume 213, Issue 1, pages 88–97, October 2007 |
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k*****n
发帖数: 417 | 35 Their top5 highly cited research papers:
Wei Gu:
Title: Negative control of p53 by Sir2 alpha promotes cell survival under
stress
Times Cited: 822 (from Web of Science)
Title: Deacetylation of p53 modulates its effect on cell growth and
apoptosis
Times Cited: 331 (from Web of Science)
Title: Deubiquitination of p53 by HAUSP is an important pathway for p53
stabilization
Times Cited: 319 (from Web of Science)
Title: Mono-versus polyubiquitination: Differential control of p53 fate by
Mdm2
Times Ci... 阅读全帖 |
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E**********1
发帖数: 73 | 36 请问一下各位都知道哪些方法可以提高胞内Ca2+浓度?我用的是MEFs.
我现在只知道一种是用50mM的KCl加到culture medium里面，induce depolarization,
然后使voltage-dependent calcium channel打开，使胞内Ca2+升高。但是有一点不确
定的是MEFs是否有voltage-dependent calcium channel。
求各位指点，在此先谢过了！~ |
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n***w
发帖数: 2405 | 37 add ATP or adenosine.
It will activate adenosine receptor, converting PIP2 to IP3 which binds to
IP3 receptor on ER to liberate calcium. |
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E**********1
发帖数: 73 | 38
Thanks a lot!
I am wondering whether it is a general method, which means this method also
works on MEFs.
And I need more details about the method you mentioned, so could you give me
a link of the protocol or a paper where the method has been used. |
|
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s******y
发帖数: 28562 | 40 大赞
up
calcium
the methods section. But they did |
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E**********1
发帖数: 73 | 41 Really thank you!
Sorry about my misunderstanding. I really don't know much about calcium.
You mentioned use aequorin as a calcium indicator. My question is how about
Fura2-AM. What are the pros and cons of these two? After the treatment, I
will do immunoflurescence staining, I am thinking if I use Fura2-AM, I can
get calcium level and localization of my target protein at the same time.
Really thanks for you time!
up
calcium
the methods section. But they did |
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n***w
发帖数: 2405 | 42 I do not know much about Fura2-AM. I read papers that use this dye
in neurons but not in MEFs.
Based on your objective, I do think probably using a dye may be better than
aequorin. But the experimental conditions need to be carefully taken care of
since Fura2-AM imaging may be affected by many variables.
Good luck.
about |
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E**********1
发帖数: 73 | 43
than
of
Thanks a ton, bro! |
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c********b
发帖数: 363 | 44 The example is still not clear: how could a phosphorylated protein with DNA
binding capacity transported to cytoplasm for degradation without further
modifications?
Second, if it is so difficult to find some examples, what is the general
significance of the potential example(s). |
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s********n
发帖数: 2939 | 45 18个cysteine，估计有不少的disulfide bonds，你是表达在E. coli的cytoplasm？如
果是的话，misfold的可能性很大。GST增加了fusion的可溶性，切掉以后就沉淀了。 |
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a********k
发帖数: 2273 | 46 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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Z******5
发帖数: 435 | 47 NE-PER® Nuclear and Cytoplasmic Extraction Reagents
Thermo （Pierce）的，货号78833或78835 |
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n***w
发帖数: 2405 | 48 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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j****x
发帖数: 1704 | 49 这个nuclear transcription factor是相对于mitochondrial transcription factor和
cytoplasmic transcription factor而言的，所以显然不能等同于transcription
factor
倒是nuclear factor和nuclear transcription factor广义上应该是一回事 |
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c********r
发帖数: 189 | 50
Lysis buffer 绝对是对的，我用了好几种，而且都在显微镜下观察了。老是在
cytoplasmic fraction 一大堆的H3，太让我伤心了~ |
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