w******e
发帖数: 1187 | 1 1st of all, I actually don't do peptide aptamer myself, so don't trust
me too much:) following is the impression I got from reading.
afaik, the peptide w/ best kd is some RGD variant--there are a loooot
of variants, such as dimer/multimer of RGD, cyclic RGD, peptidomimetic
RGD, etc. It seems to me that no linear peptides has very good kd (again
from very small sample size), since peptide chain is too flexible.
circulirization/stapling is believed to increase kd by confining the
possible conforma |
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t*******k
发帖数: 87 | 2 或者，有没有人看到文献提到ubiquitin经过100C加热会dimerize呢？
抱歉，问题太专业了，和本版气氛不太和谐， 呵呵 |
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O******e
发帖数: 4845 | 3 How did you purify your oligomeric ubiquitin? No contamination of dimers or
higher forms? |
|
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C*********m
发帖数: 213 | 5 leucine zipper?
which
these
any |
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s******y
发帖数: 28562 | 6 That is possible, but can you suggest a particular leucine zipper (name or
sequence) in particular?
I feel embarassed because I don't remember anyone of them right now. |
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l****y
发帖数: 398 | 7 GCN4
more controllable-fkbp/frb |
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s******y
发帖数: 28562 | 8 Wow, the fkbp/frb seems like a cool stuff with super high affinity!
I am definitly going to read a lot more of the literature about it. The only concern I have is those domains are bigger than my expectation.
The GCN leucine zipper sounds like a good idea because it is smaller, but the affinity is so so...let me see if they have mutants with higher affinity... |
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w******e
发帖数: 1187 | 9 conjugate to ssDNA/RNA using like NHS chemistry?
which
these
any |
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s******y
发帖数: 28562 | 10 Unfortunately we cannot do that either because we need to express the
protein in vivo.
Conjugate them with nucleotide can only be done in vitro and there is no
guarantee where the connection site is. |
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y******8
发帖数: 1764 | 11 how about Protein G B1 domain and binding motif on Fc |
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y******8
发帖数: 1764 | 12 Don't know the size limits. If the fusion domain can go above 25kd, then GST
is a very good choice. |
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y******8
发帖数: 1764 | 13 Tandem repeats of motif could increase the affinity by 10 fold or even more.
Homodimerization could be diminished by polycistronic expression.
By the way, if you are willing to dig the bacterial protein database, there
are many many possibilities. In general, those proteins are small and
readily folded. The affinity of those interaction are tremendously high. |
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y******8
发帖数: 1764 | 14 I think people hold patents on all of these. You might need to get approval
first. |
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s******y
发帖数: 28562 | 15 唉，我就是不知道该到哪里去找这种数据库啊？
我不管是什么来源的蛋白，只要那个蛋白不会杀死表达的动物细胞就好了。
有什么建议怎么去查这种数据库？
more.
there |
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s******y
发帖数: 28562 | 16 啊呀这么麻烦啊？难道这种类似的实验以前没有人设计过么？
approval |
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y******8
发帖数: 1764 | 17 The protein G B1 domain is about 8kd, and would be hard to synthesize.
Pratically, this is the only reason you need to ask for the material and
permission to start. Otherwise, you could just do it and wait for response.
Usually, people don't pay attention to nonprofit stuff. |
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y******8
发帖数: 1764 | 18 Neither do I. But very often, I am amazed by those discoveries from
microbiologists. |
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s******y
发帖数: 28562 | 19 OK... that could be quite a hassle then.
. |
|
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s******y
发帖数: 28562 | 21 小声地再问一句：
没有人知道么？
我其实要找的就是两个有比较高结合能力的蛋白domain，最好是长度都
不超过50个氨基酸的，而且序列已知的，而且是heterodimer.
which |
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d******1
发帖数: 709 | 22 Scaffold protein?
Like PDZ domain in neuron junction? two PDZ domains from two differnt
proteins pull them together to perform the function.
Zhang Mingjie in HKUST studied a lot on PDZ and PDZ heterdimerization. you
may check his website.
http://bcz102.ust.hk/index_publications.htm |
|
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c****l
发帖数: 1086 | 24 why not. check out sybr green. make sure no non-specific band or primer
dimer which will interfere with your Q-PCR readout. Usually the Q-PCR
machine will do a dissociation curve. |
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R****n
发帖数: 708 | 25 小峰温度是多少？貌似primer dimer.提高点hybridization temperature试试 |
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P******m
发帖数: 21 | 26 应该不是primer dimer, 因为标准品的曲线没有小峰。 |
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n**********s
发帖数: 14 | 27 有可能DNA AMOUNT高一些, 如果RNA量相对低的话, 有出PRIMER DIMER的可能...
RUN A GEL +1 |
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i********e
发帖数: 50 | 28 有个超大蛋白，单体为550Kda, 细胞里是tetramer。想看一下转基因表达后的状态，是
monomer/dimer/tetramer.请问除了native page还有什么方法能够鉴别或者比较
monomer/tetramer？
native page用了basic-native gel protocol。做western blot的话，使用的抗体和
monomer或tetramer结合的效果会有差异吗？比如monomer的结合效果好，band清晰。抗
体和tetramer 反应弱，看不大出来band?
比较奇怪的是，6％的胶跑3个小时和6个小时的结果不一样。难道6个小时后所有的蛋白
跑出去了？不应该啊.
求高人指点。 |
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M*****e
发帖数: 279 | 29 Baltimore does not have that many papers with >1K citations (Web of Science).
Citations of David Baltimore
1. Title: An essential role for NF-kappa B in preventing TNF-alpha-induced
cell death
Author(s): Beg, AA; Baltimore, D
Source: SCIENCE Volume: 274 Issue: 5288 Pages: 782-784 Published:
NOV 1 1996
Times Cited: 2,067
2. Title: A NEW DNA-BINDING AND DIMERIZATION MOTIF IN IMMUNOGLOBULIN
ENHANCER BINDING, DAUGHTERLESS, MYOD, AND MYC PROTEINS
Author(s): MURRE, C; MCCAW, PS; BALTIMORE |
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h*****t
发帖数: 1226 | 30 redcuing gel? if so, you will not see dimer, maybe it is just a contaminant |
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g*********5
发帖数: 2533 | 31 if you don't add dtt in loading buffer,
you could see dimer.
I AM doing this experiment now... |
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C*******e
发帖数: 4348 | 32 跑个胶看看
80度和86度还是有区别的哈
有可能是primer dimer
也有可能就是一点点contamination
realtime是比较敏感的
Ct>33的很有可能是contamination
或者样品里gDNA没除干净
或者加样的时候“飞沫污染”，比如你用的一整块96孔板，不是strip
没有办法先把无模板对照先盖起来
然后加有模板的样品的时候有那么一点点“飞沫”进去了
或者你用的不是带filter的tip
然后加样枪就受到少许污染 |
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w*****3
发帖数: 1582 | 33 cross-link, or native gel |
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h****k
发帖数: 182 | 34 谢ls,cross-link的具体思路是怎样的? |
|
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y******8
发帖数: 1764 | 36 内源的膜蛋白可能很难做。比较常见的是做FRET或者Co-IP，都是过量表达。 |
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m*********7
发帖数: 5207 | 37 Bingo! I like this answer. |
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z********i
发帖数: 610 | 38 1.看到有帖子说细胞污染的事情，我也有遇到了很奇怪的事情。我的细胞养时间长了以
后，比如2-3周，形态会发生很大的变化。比如Hep3B会变成293T那样的形态，不知道是
不是因为细胞污染的缘故？
2.我手上有一个蛋白,当我用GFP-tag去看定位，发现呈现点状定位。如果用FLAG-tag则
是弥散的（没有内源的抗体）是不是因为GFP dimerization影响了它的定位呢，或者是
因为TAG太大的原因？
谢了 |
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s******y
发帖数: 28562 | 39 Yes, because those rRNA would not form dimer with each other |
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i******m
发帖数: 495 | 40 yes, you can. just higher primer dimer and non specificity |
|
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j****x
发帖数: 1704 | 42 我们也想到过可能是dimer或者双环套叠，因为迁移速率刚好是超螺旋质粒的2倍，但是
不太懂这个在复制过程中是怎么形成的，而且条带很单一很纯，如果是套叠感觉应该是
mixture才对。
nicked plasmid可能性也不大，首先是新置备的质粒，而且胶上也能在主带后看到微弱
的nicked条带。如果主带是nicked plasmid，大小也不对（和其他衍生质粒的nicked
条带对不上）。
gDNA的污染可以排除，因为可以被完美的酶切鉴定。
确实很奇怪，有谁也遇到过类似情况吗？感觉双环套叠的可能性最大，能解释一切现象
，但是不明白怎么形成的。 |
|
k****k
发帖数: 36 | 43 你好像是不希望a同betagamma dimer分离？只要在没有receptor和gtp的条件下，a和
betagamma就应该是比较紧密的结合在一起的。 |
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c**i
发帖数: 6973 | 44 Peter Landers, New Breast Cancer Drug Found Deep in the Sea. Wall Street
Journal, Jan. 4, 2010.
http://online.wsj.com/article/SB1000142405274870411150457605977
("A study found it [Halaven] extended the life of patients with advanced
breast cancer by about two-and-1/2 months, to 13 months")
My comment:
(a) Eisai Co., Ltd. エーザイ株式会社
(a public company; headquarters Tokyo; established in 1941 by Toyoji NAITO
内藤 豊次 as Nohon Eisai Co., Ltd. 日本衛材（衛生材料の略。具体的には絆創膏
や包帯の事))
ja.wikipedia.org
(b) eribulin
htt... 阅读全帖 |
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m**********d
发帖数: 137 | 45 新设计了primer，扩增genomic DNA的某片段，170bp左右，用qPCR来定量，primer退火
温度已测好，57°C
用SYBR green做qPCR，SYBR新从BioRad订的
25μl total volume
12.5μl SYBR green master mix + 0.1μl primerF (0.1μM in the fianl
concentration)+ 0.1μl primerR + 50ng genomic DNA + H2O fill up to 25μl
用的PCR program如下：
95°C 10min + (95°C 30sec, 57°C 30sec, 72°C 30sec) 40cycle + dissociation
curve
72°C elongation step读数
结果没有任何信号，dissociation curve当然也没信号或者有一些非常弱且杂乱的信号
我把sample拿出来，跑了个2%agar gel，结果非常清晰干净的一条带，size完全正确，
看不到primer dimer或者其他non-specific ... 阅读全帖 |
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z***q
发帖数: 907 | 46 一个小的hairpin loop(27nt),用核磁做其结构。在水中看base pair(imino-imino,
imino-amino),folding correctly, connectivity也很多，但是5-8ppm的peaks are
totally
messed up, very broad and not well resolved.换到D2O中也是一样，即使2D
expperiemnts, 也不能很好的区分各个峰。1D上就像一个大的馒头峰，上面插着若干个
小叉子
（小尖峰）一样，无法分开各个峰。
像请问各位大侠，有人遇到过这种情况么？这个RNA 到底有没有正确折叠呢？从非变性
胶上只
看到一条清晰的条带，确定是monomer而不是dimer.如果结构不对的话，有什么方法可
以使其
正确折叠呢？我已经试过了加热快速冷却，缓慢冷却，改变盐离子浓度（50mM,100mM),
pH值
（试过4.5，6.0）都不管用，而且每次得到的谱图（1D）都不一样，都快被它搞崩溃了
。是不
是接下去只能考虑换序列了？老板也完全不能理解这种情况，只是反复和我强调我的操
作有错
误，他这么多... 阅读全帖 |
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x*****o
发帖数: 441 | 47 不是太懂NMR,不知道你的核磁结果的解释是什么.
你的hairpin stem是不是GC rich,会不会没有闭合.你的Tm是多少?
还有non-denaturing gel 怎么确定不是dimer? |
|
z***q
发帖数: 907 | 48 谢谢回复！
结果的解释就是所有supposed base pairs都形成了，hairpin stem有9个base pairs,
只有2个GC，Tm没测过，算出来的是54C，自由能也很低，因为序列开始是2个UU base
pair，可能不太稳定，但是从核磁上看UU base pair都形成了，所以感觉很矛盾。试过
端基变成GC,然后就发现了很多dimer.non-denaturing gel上是根据别的已知样品的对
照。 |
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r********d
发帖数: 58 | 49 盐浓度越高，越有利于稳定dimer,trimer等结构
盐浓度低一点对single hairpin好
你干脆别加盐了，如果buffer是用phosphate来控制pH的话，那么加5mM的phosphate就
可以了
你sample concentration是多少？ |
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r********d
发帖数: 58 | 50 在结构末端多加两个basepair,把结构稳定下来
同时把盐脱掉，防止dimer的形成
再就是做NMR之前用heat and fast cool,不过个人感觉这个没啥帮助
了。 |
|
