C******2
发帖数: 97 | 1
vectors
The construction is modified and derived from pRetrosuper which is used for
random mutation in host genome based on gene-trap. Truncated 3’LTR
fragment is promoter activaty lost. The idea is host genome will be mutated
by insertion because the existence of splicing acceptor(SA) fragment thus
generate a fusion RNA containing cherry CDS. Fusion florescence protein can
be detected in some case when insertion occurs as an in frame manner. The
insertion will also make the infected cells be... 阅读全帖 |
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b****r
发帖数: 17995 | 4 比如其中一个data是一种dyskinesia，就是某种异常神经放电造成的疾病
遗传方面的东西还是不会有什么问题的。是个大家系里先连锁再测序发现的，LOD>3。
Case control和GWAS的data那确实很多都难说，我目前没有打算往那方面做
这个突变是truncating mutation，造成premature stop codon，因为在外周血不表达
，还不知道会不会non sense mediated decay。ESP6500和1000Genome 都没有。而且老
鼠KO也有一点类似表型，in vitro也有几篇JBC之类的文章证实和sodium channel互相
作用
其实如果能再发现个家系，直接就nature genetics或者AJHG了，只是好家系这东东像
你说的，再找个真不容易，万一让人家抢先了可惜，能加点功能的先发了也好 |
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i***l
发帖数: 1656 | 5 如果没法做实验，非要硬凑 也不是不能
1. 蛋白Sequence Alignment,Phylogenetic Tree
2.你的实验结果
3. 找找他们各自的Homolog，有没有已知的结构，做MolecularReplacement推测结构，
Docking， ETC， 预测可能的结合位点。
看看能不能发个神马Open Access的杂志
其实 严谨的说，的确至少要做个体外实验，突变或TRUNCATE 之后不结合才能下结论。 |
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l****j
发帖数: 70 | 7 从mRNA水平看确实是基因被敲除了，这个蛋白质影响的下游蛋白质抗体比较好使，能看
出明显差异来。
truncated蛋白怎么解释？ |
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d***s
发帖数: 1062 | 8 我们有个conditional KO小鼠，敲掉了部分exon。蛋白还能表达，但是truncated的。 |
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x******3
发帖数: 111 | 9 现在正在做WB 检测GFP-Tagged的目的蛋白的表达，尝试了6种GFP 抗体，发现在人的细
胞中都能够检测到GFP 表达，但是在鼠的细胞中只有ABCAM的AB13970检测到GFP 表达（
但有其他一堆杂带），其他的抗体都能看到一堆bands，唯独没有GFP 的bands，主要是
砸带太多，曝光不能爆太久。
有大神能帮忙介绍一下在小鼠细胞中WB检测GFP 表达的经验吗?
不能用目的蛋白的抗体因为做的目的蛋白的truncated protein。
那个abcam的ab13970是鸡抗体，不知道怎么样去优化？（4%BSA block， 400mA/2hrs
transfer）
非常感谢！ |
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m******5
发帖数: 1383 | 10 同意！！
另外有一点，我觉得很多人都觉得上下两条带是alternative translation，其实很可
能是其它Sumo或者Ubiquitin或者磷酸化修饰导致的。alternative translation的可能
性我认为不大。
另一种更常见的情况是下面那条带其实是N-termianl truncation band.
我认为这几种情况都比alternative translation可能性大得多。
在楼主的case里，甚至有可能上下两条带都是Full-Length，只不过Flag antibody
affinity比HA antibody高很多，导致楼主误以为下面没有HA tag.
很多蛋白都有multiple in frame的ATG,通常后面的ATG是不会起作用的(除非有
alternative splicing或者复杂的RNA secondary structure.)
退一万步说，除了in frame的ATG,还有很多非in frame的ATG，设想一下，如果这些ATG
能起作用，细胞早就被junk protein淹没了(当然，非in frame产物如果不在最后的
... 阅读全帖 |
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g*********3
发帖数: 177 | 11 Just my opinion/impression:
1) How to define "TCA is inhibited":
Sometimes it is not totally inhibited in that whole TCA Cycle. It has
another name: Truncated TCA cycle.
2) as xhzhou said: the intermediate products in glycolysis can enter into
other pathways---"whatever pathways"---synthesized into "macromolecules".
The "whatever pathways" & "macromolecules" are hot filed, which is more
complex than the textbook. Many of them are not discovered yet and may be
tumor-specific SUCH as the oncometab... 阅读全帖 |
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B******o
发帖数: 496 | 12 用truncated蛋白制备抗体，回来以后过全长蛋白的柱子把抗体纯化一遍？
one.
in |
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l***i
发帖数: 36 | 13 感谢回复，首先这个蛋白在细胞内，因此用truncated isoform 免疫后再筛选或许会更
有效一些。正如您所说的时间应该花在科研上而不是创造实验条件上，如果您知道有什
么公司可以提供一条龙服务，可否提供相关信息。Thanks. |
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h*******o
发帖数: 4884 | 14 不清楚，不过我看我买的抗体上一般都是用peptide 来做抗原
你可以把truncated 的缺失的那一部分去做抗原吧 |
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i******1
发帖数: 21 | 15 1. RNA level check: 1) to make sure the real-time rt-PCR resulst are
specific, using >=2 unique sequences of your target transcript in the
presence of proper controls than unambiguously tell quantification accuracy
and amplification specificity. 2) see how many isoforms in your cell line;
are they in similar lengths; do they share the sequence that can be targeted
by your all your siRNAs? is it possible that the one that cannot be knocked
down have predominant abundance and it it functionally ne... 阅读全帖 |
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j***x
发帖数: 1469 | 16 Virology. 1993 Dec;197(2):751-6.
A truncated form of herpes simplex virus type 1 immediate-early protein
Vmw110 is expressed in a cell type dependent manner.
Everett RD1, Cross A, Orr A.
Please send to : [email protected]
/* */
ThX |
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w***r
发帖数: 709 | 17 1. Nature. 2002 Jan 3;415(6867):45-53.
p53 mutant mice that display early ageing-associated phenotypes.
Tyner SD(1), Venkatachalam S, Choi J, Jones S, Ghebranious N, Igelmann H, Lu
X,
Soron G, Cooper B, Brayton C, Park SH, Thompson T, Karsenty G, Bradley A,
Donehower LA.
Author information:
(1)Cell and Molecular Biology Program, Baylor College of Medicine, Houston,
TX
77030, USA.
Comment in
Nature. 2002 Jan 3;415(6867):26-7.
The p53 tumour suppressor is activated by numerous stressors to ind... 阅读全帖 |
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e****s
发帖数: 1125 | 18 你这要是in vitro的蛋白试验，才可以把位置的迁移归结于磷酸化。
要是在Cell line里面的话，就不好说了。上面说的Truncated 应该是可能的。 |
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C*********m
发帖数: 213 | 19 It's an ER-resident protein. According to the literature, upon kinase
stimulation, it goes inside the nucleus. We did see nucleus localization for
a slightly-truncated version. We have a hypothesis how this happens based
on other data we have, and is confirming this using cell-based assay. Would
not reveal identity of this protein for now, but it is up-regulated in two
types of endocrinological cancers. |
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j****x
发帖数: 1704 | 20 之前没留意过，近期突然发现，Clontech家的所有Lentiviral vector系统，包括其经
典的Tet-on/off/one lentivector，尽管宣传是最安全的第四代载体系统，但其中的
HIV-1 5'/3' LTR居然都是wildtype的而并非是被几乎所有其他商用载体所一致采用的
truncated/∆U3 LTR，作为self-inactivating (SIN) vector。这是件很奇怪的
事情，有哪位对Lentiviral vector比较熟悉的能讲讲吗？谢谢 |
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l******a
发帖数: 3339 | 23 HA-Nter clone 和 HA-Cter clone在同一个vector上？两个promotor，还是怎么做的？
ter |
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r******g
发帖数: 600 | 24 sorry，没有讲明白
2个 独立plasmids,都是retroviral plasmids
promotor是 CMV
最后克隆的两个plasmids
HA-Nter
CMV promoter--Multicloning sites--- HA tag- N-terimus+Transmembrane domain+a
few more C-ter nucleotides----- IRES------EGFP
HA-Cter
CMV promoter--Multicloning sites--- HA tag- Transmembrane domain+C-terminus-
---- IRES------EGFP |
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e****s
发帖数: 1125 | 25 Double check 你的克隆。Make sure stop code是正确的，没有产生Frame shift.
我觉得最可能的解释就是Frame shift，所以你的蛋白和Tag都在，但在后段可能有
Frame shift突变，把Stop code给突变了。所以一直表达到70K，遇见另一个Stop code
再停下来。
+a
terminus- |
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r******g
发帖数: 600 | 26 这个是 我第一个检查的～ doublecheck了很多遍
没问题～
westernblot上面 很明显的，有一个 正常molecular weight的 很浅的lower band，
然后一个很粗的 upper 70kd的band
因为，有lower band，所以，也不大可能是 frameshift，不然 根本不可能有lower
band，对不对？ （293 不表达endogenous的这个 protein）
code |
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n***y
发帖数: 114 | 28 1. 给插入的蛋白和GFP换个位置。
2. 用PP2A。 |
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W*V
发帖数: 13 | 29 using loading buffer containing 4M urea + 2mM 2-mercaptoethanol. If you get
right, please feedback. |
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r******g
发帖数: 600 | 30 I will give a try first thing next week. Thanks
You mean loading for Western blot right?
get |
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W*V
发帖数: 13 | 31 Right. loading samples on gel runing SDS-PAGE. |
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i***l
发帖数: 1656 | 32 agree with above,maybe oligomer due to non specific hydrophobic interactions
, which is not uncommon among transmembrane proteins.
4-6M urea, try to completely denature your sample,
however, if oligomer forms, sample usually runs in a ladder pattern, say,
dimer, tetramer, etc. if you only see two major bands, one monomer, the
other ~4x or 6x apparent molecular weight, it's still a little weird to me. |
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M******g
发帖数: 152 | 33 Try not to heat up your protein samples in SDS loading buffer. Many
membrane proteins form aggregates even in SDS-loading buffer when they are
heated. 70 kd may be your protein aggregation. |
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M******g
发帖数: 152 | 34 Try not to heat up your protein samples in SDS loading buffer. Many
membrane proteins form aggregates even in SDS-loading buffer when they are
heated. 70 kd may be your protein aggregation. |
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p****t
发帖数: 18 | 35 可能抗体不特异，楼主可以把70kDa的条带切下来送质谱。
如果是oligomer，质谱也能看出来。
ter |
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v********a
发帖数: 646 | 36 面对这个1979年的单身女郎，从中专毕业，到参加成人自考拿到同等学历，然后一路逆
袭，最后傲然在苏州大学就职。
只有两个字：佩服
http://hlxy.suda.edu.cn/jiaoxuekeyan/yanjiushengdaoshiminglu/20
王丽，女，教授，硕士研究生导师
研究方向
慢性病运动康复：重症病者包括心脑血管疾病、骨关节系统疾病和终末期肾病等患者的
运动康复。
体能与疾病：体能水平与疾病发生和预后之间的关系；
社区康复：糖尿病、骨质疏松等的社区康复护理干预。
联系方式
电话：0512-65221499 (办公室); 138-1486-1702 (手机)
邮a class="__cf_email__" href="/cdn-cgi/l/email-protection" data-cfemail="3dd99e875154104a5c535a100c7d4e48595c13585948135e53">[email protected]/* */; [email protected]/* */
地址：苏州市十梓街1号苏州大学护理学院本部20... 阅读全帖 |
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发帖数: 1 | 37 这个intron后面还有11个exon，应该是产生truncated mRNA吧
CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCC
CACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGG |
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m*********D
发帖数: 1727 | 38 开始也这么想。其实和你突变的地方有一定关系。在基因的起始部位，Knockout比较容
易，在靠近结尾的地方，你会有truncated蛋白表达。这种的活性是无法预料的，如果
是dimer，就更难了。另外，压迫看protein的表达量，靠近结尾的，往往没有合适的
antibody来区分你要的replacement和trancated copy。 |
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l********e
发帖数: 415 | 39 有道理。对于无法筛选的基因，你觉得做两轮targeting怎么样。先拿杂合，然后
target杂合拿纯合。
: 开始也这么想。其实和你突变的地方有一定关系。在基因的起始部位，Knockout
比较容
: 易，在靠近结尾的地方，你会有truncated蛋白表达。这种的活性是无法预料的
，如果
: 是dimer，就更难了。另外，压迫看protein的表达量，靠近结尾的，往往没有合
适的
: antibody来区分你要的replacement和trancated copy。
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a****o
发帖数: 6612 | 40 There is only ONE full CI, no matter you are starting from one reference
(like HF determinant) or multi-reference (like MCSCF active space).
single or multi-references only matter when you have a truncated
excitation level.
CASSCF
excited |
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n********r
发帖数: 195 | 41 没错，project meeting的从safety accessment到clinic PK，从pharmacology到
analytical都要参加的，你光知道个push botton的话坐那里跟sb一样。最简单的，人
家做poppk的人问你，你的数据出来truncate到小数点后几位你能答上来吗？ |
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t****n
发帖数: 39 | 42 The order of algorithm means the order of your truncation error. Say a
second order algorithm means the error is O(dx^2), where dx is your step size.
So a higher order algorithm does not necessarily give out more accurate
solution than a lower order one. |
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m******o
发帖数: 2 | 43 It depends on the problem you are solving. If the partial differential
equation itself is unstable (say turbulence, molecular dynamics), then even
the very tiny difference of machine truncation errors will eventually give you
very different answers. However, the statistical quantities(such as
correlation coefficients) should remain the same.
编译 |
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w**d
发帖数: 2334 | 44 it depends on the scheme. Usually the diffusion is determined by
the leading even term of truncation error. |
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f****r
发帖数: 27 | 45 It's called truncation error |
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g******s
发帖数: 733 | 46 i don't think it's truncation error. Even using double precision, the error
doesnot change. I believe sth is wrong in the codes or somewhere. |
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o****o
发帖数: 8077 | 47 要做一个很简单的数值积分问题，其实就是求如下一个简单幂函数的期望
F(U)=U^(a-1)， 实数 a > 0。
U的分布是一个Normal(\mu, \sigma) truncated at 0.
在对数据集中每个点进行这个计算的时候，出现了很奇怪的问题。我用的是quad，高斯
quadrature法
当abs(\mu) 远离0点， \sigma又较小的时候，即使\mu1 \mu2很接近计算出来的值也相差
很大。例如
>> quad(@numF1_e,1e-10,1-1e-10,0.00001,0, 3.0692,1.2014,0.5740,9.389589)
ans =
139.9154
>> quad(@numF1_e,1e-10,1-1e-10,0.00001,0, 3.0692,1.2014,0.5740,9.789589)
ans =
2.5851e-005
>> quad(@numF1_e,1e-10,1-1e-10,0.00001,0, 3.0692,1.2014,0.5740,9.689589)
ans =
149.5704
@numF1_e 是那个函数 |
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O******e
发帖数: 734 | 48 If you are using a Fortran 9x compiler, see what this gives you:
print*,selected_real_kind(33,4931)
If you get a positive integer, your compiler claims to support
IEEE quad-precision real variables. If you get a negative number,
your compiler does not provide built-in support for QP.
Intel's ifort supports QP by emulation and provides QP versions
of the intrinsic functions. There is one bug that I know of in
the way QP numbers are printed--they are truncated and printed
as DP numbers, but th |
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f*******a
发帖数: 80 | 49 Plane wave simulation is an open boundary problem. You need perfectly
matched layer (PML) to truncate your computation domain. PML can be
implemented in finite element or finite difference time domain method.
边界 |
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w*******U
发帖数: 256 | 50 用 gfortran 编译，
gfortran -o test test.f
结果报错
relocation truncated to fit
应该是stack的内存小于2G的问题，然后加上选项
gfortran -o test test.f -mcmodel=medium
结果没有报错，但有 warning
Warning: ignoring incorrect section type for .lbss
也生成了可执行代码，试运行了几步，也还行。但是不知道这样的 warning 会不会有
什么潜在的危险？
谢谢大家！ |
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