w******e
发帖数: 1187 | 1 非常感谢！再问个相关的问题：human protein有多少percentage是没有homologue（
across all organisms)的？ |
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l**********1
发帖数: 5204 | 2 0.5% human protein is no homology to Chimpanzee
five years ago that value was 0.9 %. |
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y*******a
发帖数: 303 | 3 如题，已知一个基因的信息，prediction是Serine/threonine-protein kinase，但不
知道substrate，怎样从实验的角度证明是protein kinase? |
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y*******a
发帖数: 303 | 4 可是我的这个protein kinase gene 是新的，目前data base上已经可以得到cDNA, 但
是protein还没得到，是不是就没有办法缩小范围了呢？ |
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b******y
发帖数: 627 | 5 Some kinases auto-phosphorylate. If you eventually get protein, either
through recombination or IP, try to incubate it at high concentration in the
presence of p32-ATP and Mg2+ and auto-radiography to see whether your
protein auto-phosphorylate itself.
However, bioinformatics are pretty powerful. If it says that it is S/T
kinase with high confidence, it is. However, substrates of kinase are non-
trivial to find for three reasons: 1) interactions between kinases and their
substrate are mostly tra... 阅读全帖 |
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y*******a
发帖数: 303 | 6 如题，已知一个基因的信息，prediction是Serine/threonine-protein kinase，但不
知道substrate，怎样从实验的角度证明是protein kinase? |
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y*******a
发帖数: 303 | 7 可是我的这个protein kinase gene 是新的，目前data base上已经可以得到cDNA, 但
是protein还没得到，是不是就没有办法缩小范围了呢？ |
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b******y
发帖数: 627 | 8 Some kinases auto-phosphorylate. If you eventually get protein, either
through recombination or IP, try to incubate it at high concentration in the
presence of p32-ATP and Mg2+ and auto-radiography to see whether your
protein auto-phosphorylate itself.
However, bioinformatics are pretty powerful. If it says that it is S/T
kinase with high confidence, it is. However, substrates of kinase are non-
trivial to find for three reasons: 1) interactions between kinases and their
substrate are mostly tra... 阅读全帖 |
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e***o
发帖数: 344 | 9 不知道这套系统的效率如何。RNA-Protein复合体需要进入核内才能激活报告基因。不
知道怎么才能高效入核激活报告基因。和RNA IP不知道哪个更好。好像现在有利用RNA-
Protein 交链的方法，但不是很明白。 |
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c**********o
发帖数: 69 | 10 NaCl : shielding effects/stablize protein
site |
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t**m
发帖数: 158 | 11 structrual biology 入门,
推荐"Introduction to protein structure" by Carl Branden & John Tooze
site |
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h*********c
发帖数: 78 | 12 protein structure and function, 灰色封皮的,非常适合新手入门 |
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x********u
发帖数: 430 | 13 I have been running SDS-PAGE several times and cannot get clear band for low
molecular proteins. Any suggestions will be highly appreciated.
Attached is a gel picture I just took yesterday. (12% resolving gel and load
50 ug total E. coli cell lysate proteins). |
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i******w
发帖数: 407 | 14 用的是Qproteome Bacterial Protein Prep Kit，里面有Benzonase Nuclease，结果跑
SDS-PAGE然后coomassie染色后，总在最下面几kb左右出现一条超蓝的带。
直接用cell culture加laemmli buffer跑就没有那条带。所以我猜应该是Nuclease。
请问一下谁知道怎么能把protein extract里的Nuclease去掉啊？谢谢 |
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w*********n
发帖数: 439 | 15 各位大虾，我是新手，正准备开始做拿小鼠T cell作 CHIP assay。
请问你们都是用的那个公司的protein A/G beads？ protein A 和G 该选择那个？
非常感谢~ |
|
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e****e
发帖数: 1042 | 17 Thanks for your suggestion! But we currently already use HPLC to quantify
our peptide product. Protein cannot be quantified by FPLC\HPLC, right? I'm
not sure about Mass spec though. |
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e****e
发帖数: 1042 | 18 No. I came from biochemistry labs. So can I get an conclusion that there is
no a method can detect only the amount of protein but peptide? Of course I
have a method to detect the amount of the peptide product. |
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s******y
发帖数: 28562 | 19 我明白你的意思了。
你本来就是想看看有什么最新的特别fancy 的技术可以不通过分离而直接
挑出peptide or protein来特异反应啊。
那还真的没有这种技术。
因为蛋白本来就和peptide 没有啥严格区别。
is |
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b******y
发帖数: 627 | 20 This depends on what peptides you are talking about. Over the years, I have
produced multiple peptides (some are qualified as proteins, e.g. ww domains)
between 3-5 KDa in E. coli: some of them are disordered, some are single
helix and some are a few beta-strand. Many of them have very high yields. |
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n********k
发帖数: 2818 | 21 hi, buddy, what define a protein or peptide, is it the size? 50-100aa?
have
domains) |
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s*******p
发帖数: 41 | 22 Did anybody try protein structure service company? I am working on a
mutant of well studied protein. An insight about molecular structure might
give additional support on my conclusion. Is there any company provide this
service? Thanks a lot in advance. |
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y***i
发帖数: 11639 | 23 你这个思路有点怪。什么叫被其他protein用烂了？你用的所有的技术，无论是分子克
隆还是显微镜都是被其他protein用得烂得不能再烂了。技术哪里有用烂了这一说？ |
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m******p
发帖数: 67 | 24 western for a 25kd protein. using serum.
but the band is clearly at 50kd.
seems dimer, but in sds-page, proteins are denatured, so should not form
dimmer. any suggestions?
thank you very much. |
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s******y
发帖数: 28562 | 25 有啊。
Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):11259-63.
Signal-induced degradation of I kappa B alpha requires site-specific
ubiquitination.
Scherer DC, Brockman JA, Chen Z, Maniatis T, Ballard DW.
Source
The inhibitor protein I kappa B alpha controls the nuclear import of the
transcription factor NF-kappa B. The inhibitory activity of I kappa B alpha
is regulated from the cytoplasmic compartment by signal-induced proteolysis.
Previous studies have shown that signal-dependent phosphorylation ... 阅读全帖 |
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p*****e
发帖数: 93 | 26 1.
CARMA3 deficiency abrogates G protein-coupled receptor-induced NF-κB
activation
Brian C. Grabiner1,5, Marzenna Blonska1,5, Pei-Chun Lin1, Yun You1, Donghai
Wang4, Jiyuan Sun1, Bryant G. Darnay2, Chen Dong3, and Xin Lin1,6
2.
MEKK3 is required for lysophosphatidic acid-induced NF-kappaB activation.
Sun W, Li H, Yu Y, Fan Y, Grabiner BC, Mao R, Ge N, Zhang H, Fu S, Lin X,
Yang J.
3.
Bcl10 plays a critical role in NF-κB activation induced by G protein-
coupled receptors
Donghai Wang * , † ... 阅读全帖 |
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y*******a
发帖数: 303 | 27 我最近一直在克隆一个protein kinase gene，连在T-easy vector 上还挺轻松，然后
通过粘性末端切下来， 和pCambia binary vector 相连转入E.Coli后就出现问题了。
有时候菌很难长，有时候长的size正常，做colony PCR没有，以为是gene 太长（3.5kb
）普通的Taqpolymerase很难PCR出来，所以用基因的部分片段PCR，colony PCR都有条
带。提取plasmid后酶切总是很乱，所幸去测序，但都是没测序结果。请问是不是
protein kinase的表达会影响E.coli？
非常感谢！ |
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t**l
发帖数: 109 | 28 为什么cancer cell的heat shock protein水平高，普通细胞低？
难道heat shock protein 不是好东西吗？还是搞不明白。如果普通细胞over express
一些HSP，会怎么样？
为什么细胞只有在stress的时候才会出现HSP表达高，一直高不行吗？
还有一个问题：那为什么CANCER CELL 很怕HSP inhibitors?
如果用HSP inhibitor,普通CELL是不是也跟着一起死？这种HSP inhibitor 抗癌药物有
什么特效和好处呢？还是说普通CELL，不是那么害怕HSP inhibitor? |
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f*****f
发帖数: 195 | 29 方法很多：
一种是稳定的dimer，比如二硫键形成的dimer，SDS lysis buffer 也不能破坏，
Western blot会有两条带，刚好两倍差别。一定要确定的话可以IP后可以mass-spec
二假如SDS lysis buffer 会破坏，可以先IP后，sucrose gradient或是分子量层析，
至少有2个component
三，假如蛋白能纯化出来的，就更容易了。直接GST-protein过分子筛。
四，IP或pull down用外源蛋白可以沉淀下endogenous protein，说明可以形成dimer或
polymer
五，X-ray and structural determination确定dimer结构 |
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z***g
发帖数: 28 | 30 Thank you pal, actually the flowthrough is the third left to the marker,
sorry for not mentioning...and the 23KD and 18KD are also in the flowthrough
( but so weak compared to the peak I collected), it's possible that the
protein was trapped, but the amount of proteins seems the same to me,
although we didn't quantify... haha, thank you very much for answering |
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K*F
发帖数: 120 | 31 为申请EB1-A，求审稿机会，Biochemistry, protein chemistry, protein
engineering方面，非常感谢！ |
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f*******0
发帖数: 62 | 32 因为有挺多人问这个position,我就不一一回复了。哪位觉得冒犯的还请多多包含。
我的研究主要是express and purify bacteria membrane protein. 一旦蛋白样品有了
，很多characterization can be followed, including protein crystallization.
因为我自己也是今年8月才会到新学校，很多东西都得从零开始。所以我希望跟我合作
的partner能独立的完成从买仪器，调试仪器，初步实验，优化实验。如果你觉得你能
独立完成这些，我觉得我们合作的机会还是很大的。当然我也欢迎你有自己的projects
, as long as they fall in the category of my long term goal (i.e.,
publication and funding).
--------------
平时不怎么发文，写的不够清楚还请见谅 |
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d****u
发帖数: 275 | 33 Surface/membrane protein extraction kit
1)Incubate cells with biotin
2)IP with Avidin
3)Detect your protein of interests.eg WB
Good luck. |
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d****9
发帖数: 517 | 34 有个compound, 可以激活both humoral and cell-mediated immune responses，但是
不知道机理，想做一个protein microarray for immune response (cytokine,
chemokine, DC maturation marker, etc.,)
对这个领域非常不了解，不知道protein microarray可不可行，还是必须用gene
microarray。可以推荐个microarray，给点建议吗？多谢！ |
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s******s
发帖数: 13035 | 35 条件啊，control啊，等等。你现在研究这个没意义，因为
做不出，我还在上面费过很多时间呢。还是等MS的分辨率提高
两三个数量级以上，那时候用类核酸tiling array一类的东西
抓下来就行了。或者等牛x发明pcr for protein， 或者吧protein
转化成核酸的方法也行 |
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e****s
发帖数: 1125 | 36 Your title is really confusing.
This could be very challenging. Because the truncated forms, especially
those with similar size to the full-length, behave very similar to full-
length protein. If most of your truncated is smaller than half of the full-
length, then it is doable. Gel filtration may helps to remove those big
truncated ones. You can also try those traditional columns, such as ion-
exchanged or hydrophobic columns.
Amacon's concentrator was not designed to do this.
I will suggest to... 阅读全帖 |
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s****9
发帖数: 932 | 37 版上有没有大牛知道如何用enzyme去掉细胞表面的所有的GPI－anchored protein。
我记得以前读过文献说可以用enzyme digestion to remove all the GPI－anchored
protein on the cell surface。现在怎么也找不到那个文献了。 |
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d*x
发帖数: 51 | 38 Thanks, Emases,
I did the half life of the protein, the phosphoralated protein more stable. |
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d*x
发帖数: 51 | 39 Thanks, Emases,
I did the half life of the protein, the phosphoralated protein more stable. |
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l******a
发帖数: 3339 | 40 negative control: no transfection, no target protein detected. 证明没有內源
蛋白，
positive control: any other flag tagged protein, 证明抗体ok，
最后是同一个质粒，把gfp放进去检测，以证明你的质粒系统没问题，抗体没问题
这些都做了，估计就是protease cleavage了，我觉得不会切得那么彻底，cell lysate
加protease inhibitor了吗？ |
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w********a
发帖数: 324 | 41 问一下如果想电转 protein （restriction enzyme）到gram-positive的bacteria体内
的话，应该注意些什么（跟电转DNA相比）？
比如：ptotein浓度？如何保证protein活性？等等？
谢谢 |
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f*********9
发帖数: 258 | 42 用crispr／cas9
给这个蛋白的chr上加个tag然后做RNA IP
或者用biotin label 的oligo pulldown RNA 核protein 做protein seq |
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K******S
发帖数: 10109 | 43 好奇，什么标准算high throughput protein identification？
用PROTEOMICS的方法，先FRACTION，20多个LC-MS/MS，得出成千上万个PROTEIN ID算吗？ |
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s********n
发帖数: 2939 | 44 Project在准备阶段，也不知道high throughput protein identification这个说法是
否准确。
主要是想做人或者小鼠的proteome（可以是whole or partial），chromatography/MS
当然是最有效的，只是想探讨一下其他可能性，比如protein microarray，或者其他非
MS的方法。
如有用辞不当不要见笑。
吗？ |
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w********a
发帖数: 324 | 45 正在构建CRISPR/Cas9的vector做genome Editing。可是怎么都不work。
现在想先试一下Cas9 protein的 in vitro activity （targeting on 我们的 target
system），不知道各位知不知道市场上有没有commercially available的Cas9 protein
可以订购？
谢谢！ |
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K**R
发帖数: 193 | 46 I focus on a protein. The difference of protein level between con and
treatment is significant, while mRNA level is reverse relation.
would you give me some suggestion and hint?
I totally have no idea :(
Thanks!! |
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e****p
发帖数: 354 | 47 Pull down & Mass spec - a lot of proteins 60-80
Ask for help, - how to find the protein complex that my gene interacts with? |
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s******3
发帖数: 7 | 48 The lab of Chuck Sanders at Vanderbilt University has an opening for a
postdoctoral fellow to conduct studies of the structure, folding, and
function of the human KCNQ1 potassium channel.
KCNQ1 is a critical voltage-gated channel in the cardiac action potential.
Mutations in KCNQ1 and its modulatory partner, KCNE1, result in various
cardiac arrhythmias, including long QT syndrome and sudden infant death
syndrome. Mutations can disrupt normal channel function by disrupting
folding, by altering ... 阅读全帖 |
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G********e
发帖数: 528 | 49 I'd like to ask if it is possible to do so.
For example, PFA fixation, FACS sorting for specific cell population, then
lyse protein for western.
The reason not doing live cell FACS is lack of the cell surface marker for
the specific cells I'm interested in. I have to do fixation/permeablization
and intracellular marker for cell purification. But after that, it seems
very hard to get protein extract from fixed cells. Can I use mild fixation?
Please let me know your experience, if any. |
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s******9
发帖数: 283 | 50 有人知道他刚建的Protein Design研究所？好像目标放在应用上。
如果能把binder proteins和enzyme的设计时间和成本极大的降低，就会带来生物技术
的革命。
当前蛋白设计的瓶颈是什么，功能筛选？ |
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