i*****g
发帖数: 11893 | 1 很有意思，看见这么多人在说这个想法。
这个想法我在2005年1月之前，换实验室时，和PI说了，他听不懂这张PPT意味什么。
后来我又换了实验室，2007年（2008？）某天新老板和我听完某个不相关的seminar，
和我说起这个，他才开口说了2句，我就知道他想说什么，
他的反应是，i have not finished my briefing yet, you really understand my
point? 于是就和他说，of course i know what you want to do,然后讨论了半天，原
来他的朋友有这个想法 etc etc，后来我又做了一些Homework，还是决定放弃。这个很
不好做，估计是个死途。
这个技术最大意义是：能找到locus specific DNA-protein complexes。目前的通行做
法是，已知某个protein, 你用CHIP，证明在某个Locus上面，然后开始making story，
编剧。但其实，那个Locus上面可能有几十种特殊蛋白在调控！！！
2004年底，我的想法来源于推广TAP+MS，我已知 TAP+MS... 阅读全帖 |
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a***a
发帖数: 40617 | 2 第一次用这玩意儿，大概40nt，5' cy5 label
不是说cy5是water soluble的吗，我用水和ph8的tris，感觉这玩意儿不怎么溶啊。。。
正常吗？跑胶一片smear，我悲剧了。。。 |
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s********n
发帖数: 2939 | 3 Thanks for sharing!
你有没有比较过用这种方法提的蛋白和常规方法（soluble）提的蛋白活性上有无明显
不同？ |
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z*t
发帖数: 863 | 4 Beutler得奖，Medzhitov会不会有意见？
http://www.sciencedirect.com/science/article/pii/S1074761309002
Ruslan Medzhitov
This year marks the 20th anniversary of a publication (Janeway, 1989) that
in many ways revolutionized our understanding of the immune system. As part
of the proceedings of the Cold Spring Harbor Symposium on Immune Recognition
, the paper authored by the late Charles A. Janeway, Jr. was not a standard
peer-reviewed publication. In fact, Charlie used to refer to it as “the
best paper I've ... 阅读全帖 |
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s******c
发帖数: 331 | 5 晶体学取得突破性进展才几年？一个新药的研发周期是几年？在10年前你做一个
soluble protein，比如一个重要的protease的结构，可能发Nature or Science，因为
那个时候还很难做，现在？structural genomics如火如荼的做structures也不过最近7
，8年的事，很多重要的drug target的结构解出来也不久，drug discovery一定从
protein structures中间受益很多，只是这种益处还没有大规模的表现在新药上，give
it time, you will see. |
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i*********0
发帖数: 915 | 6 in vitro 可以.
不过听说也有人直接把restriction enzyme放到细胞里面去切DNA的,因此,你的问题理
论上也可以.
不过效率高不高就不知道了. |
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l******g
发帖数: 1623 | 7 你说加Cre 酶吗, 这个很难进2层膜跑到核里面去, 还能保持活性吧. 应该作
transduction. |
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J********3
发帖数: 3151 | 8 Ad-Cre（腺病毒Cre）加到培养基里，效果还可以 |
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h***a
发帖数: 145 | 12 thinking about the same thing... |
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s********s
发帖数: 67 | 13 弄个表达cre的质粒transfection 就行 |
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y****u
发帖数: 74 | 14 各位老大，用loxP老鼠交配RosaCreERT2，然后分离细胞体外4-hydroxytamoxifen如何
？ |
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m*****u
发帖数: 15526 | 15 可以。不过太费劲和花时间。用permeable cre peptide做短平快实验完全没问题。但
最好用cre报告基因比如YFP，好知道作用效率 |
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f*******a
发帖数: 671 | 16 哈哈,我也正打算做相似的试验,培养loxp元代细胞再转表达cre的lentivirus |
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z*h
发帖数: 773 | 17 Most biofuels seemingly "big" papers just cheated laymen.
Example 1. E.J. Steen, Y. Kang, G. Bokinsky, Z. Hu, A. Schirmer, A. McClure,
S.B. del Cardayre, J.D. Keasling, Microbial production of fatty-acid-
derived fuels and chemicals from plant biomass, Nature, 463 (2010) 559-562.
This is a combination of X. Lu, H. Vora, C. Khosla, Overproduction of free
fatty acids in E. coli: Implications for biodiesel production, Metab. Eng.,
10 (2008) 333-339. and a lot of old papers about xylan-utilization.
... 阅读全帖 |
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F******y
发帖数: 1988 | 18 Clin Physiol Funct Imaging. 2002 Sep;22(5):312-9.
Decreased actin solubility observed during ATP-depletion is mimicked by
severing agents but not depolymerizing agents in isolated and cultured
proximal tubular cells.
To : f*************[email protected]
5黄包子，呵呵 |
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m******5
发帖数: 1383 | 20 The word I cited was from the paper who originally characterized DsRed
expression2, The clontech Manuel seems to have provided very different
information. I rather believe the former.
fluorescent
solubility, |
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b****s
发帖数: 148 | 21 Ab is less likely to go wrong if you can have signals from other probings.
your sample prep might be the target of troubleshooting.
1) did you freeze and thaw your sample frequently? - if yes, your current
samples are no longer reliable. make aliquotes in the future, or boil
protein right after lysis.
2) must snap freeze and thaw on ice for all ampules
3) double check your lysis process and use new protein samples -
microtubules can be very hard to dissolve or remain soluble |
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R*****o
发帖数: 12 | 22 Our mAb libraries are made with 6 or more diverse peptide/soluble-domain
antigens, and recognize 4 - 12 epitopes on your target protein. These
libraries will multiply your chances of getting successful antibodies,
propelling your research to its ideal goal.
For only USD 1,499, you will receive a library
- consists of 6 monoclonal antibodies,
- made with 6 DIVERSE peptide antigens,
- within 4 months.
All you need to provide is your protein sequence.
Become one of our more than 1,200 customers wor... 阅读全帖 |
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b******y
发帖数: 627 | 23 My guess is as following:
he/she is purifying membrane proteins (or protein complexes). Proper
interaction with detergent is required for its solubility and homogenous
behavior. Otherwise, it is death penalty for crystallization.
If using centricon or amicon, the protein sample will be overly concentrated
at the bottom of the tubes, so will be the detergent. In this case, it
might be over its critical concentration and therefore is likely to form
micelle (I don't have problem with michelle!). At... 阅读全帖 |
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r***e
发帖数: 2539 | 24 我phd做这个的，但是没听说过inhibitor，不过可以用soluble ligand竞争吧。
--------------------
update,刚才查了下，就是糖基类似物，基本上就是竞争结合。
selectin |
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z*******6
发帖数: 679 | 25 具体你说小分子inhibitor，这个我真不清楚，因为我自己不做这方面...
感觉你也不希望用抗体了，我能提供的一点点信息就是用PSGL-1的抗体或者soluble
PSGL-1之类的东西，这样selectin绝大多数的功能就被block了... 就像我之前说的，
看你要block的多大程度了... 如果一般需要，这样应该就可以了... |
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z***g
发帖数: 28 | 26 Hey guys
I am doing purification of membrane proteins to get enough concentration for
NMR study, the protein was labeled by his-tag
Firstly, the protein was expressed by E. coli, lysed by several enzymes,
agents and detergent(empigen) to solublize everything, then it was purified
by Ni-NTA column, the protein shows at the right MW(about 18KD) at commassie
blue stained gel.
However, the problem is, after the second purification---ion exchange on
Capto Q(pH 8.0), the protein which we collected doe... 阅读全帖 |
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M*******C
发帖数: 183 | 27 买的Sigma的 Cat. C3886,
说明书显示 Solubility (Color)
5 mg + 5 mL, H2O
% Dye content
它的意思是5mg 溶解在5ml 水里？ 但看到有的网页还加Ethanol.
www.springerprotocols.com/cdp/search/searchResultPage?text=crystal violet
Thanks. |
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f******g
发帖数: 382 | 28 问题１
哪个refolding screening kit比较好， 产品太多， 看花了眼.
问题2
求推荐个96-well filtration plate 做96-well solubility test.
跪谢！ |
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n***w
发帖数: 2405 | 29 you can dissolve in PBS and I think the solubility is up to 10mg/ml in PBS,
if I remember correctly. You can also check the package insert for L-NAME or
the MSDS to find such information.
I make mine at 5mg/ml in PBS and the dose is 30mg/kg body weight.
The half-time of L-NAME varies in literature but the ballpark is around
250min to 300min.
I do i.p. injection and do my experiments after 2 hours. I see an effective
lowering of Nitrate+Nitrite level. Some papers even do it after 1 hour.
If you w... 阅读全帖 |
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s******y
发帖数: 28562 | 30 一般来说。。。如果纯蛋白能溶的话，一般都是折叠正确了的。
当然也有例外。比方说如果带电量很强的话，有时候会出现所谓的soluble aggregate
，就是单独的情况下能溶解，但是一碰到什么核心立刻就纠缠起来沉了。
你这个情况听起来有点象这个情况。
既然你说了A蛋白用GST-bead的话会导致迅速aggregate, 你是否试验过把B蛋白先
cross-link 在什么bead 上，然后去结合低浓度的纯化之后的A? |
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z******i
发帖数: 120 | 31 Most likely they formed aggregate.
I had experience like that when mix A and B.
It's a headache.
Finally, I tried to fusion ed the smaller one with GST tag, and succeed.
If doesn't work, try MBP fusion. Or even try both fusioned with soluble tags.
Please let me know if you solve the problem. |
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z******i
发帖数: 120 | 32 仔细看了一下你的问题，你说的这个分别都挺好，混起来就沉淀，很常见。并非奇葩。
1.A B 是一样的BUFFER 吗？ 不是的话，把A加到B的fuffer里有沉淀不？
2. 给A 和B 全加上SOLUBLE TAG, 比如 MBP 或者更强的 NUSA tag， 不相信你的蛋白
还会沉淀。结构是HELIX BUNDLE 吗？网上预测一下。
3. 蛋白结合DNA/RNA不？ DNAse RNAse 不能除去很彻底。 要用PEI 和 硫酸铵。 另外
，高PI 很容易结合DNA/RNA, 并且在有GST存在的情况下有假结合。这个说来话长。 |
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l********n
发帖数: 260 | 33 Maternal asthma and microRNA regulation of soluble HLA-G in the airway.
Nicodemus-Johnson J, Laxman B, Stern RK, Sudi J, Tierney CN, Norwick L,
Hogarth DK, McConville JF, Naureckas ET, Sperling AI, Solway J, Krishnan JA,
Nicolae DL, White SR, Ober C.
J Allergy Clin Immunol. 2013 Mar 24. doi:pii: S0091-6749(13)00197-8. 10.1016
/j.jaci.2013.01.037. [Epub ahead of print]
please send paper to a*************[email protected]
Thanks! |
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E**********1
发帖数: 19 | 34 1. okay, first of all, I really don't know PDB actually provides predicted
3D models for every single PDB entry--maybe I am outdated. But if you could
provide everybody here with an example or a link, that would be very
helpful. I
am not sure if you are talking about homolog structures or homology
modeling. Neither is accurate information. Homology modeling is very
challenging especially when sequence homology is low. Often you cannot find
solved homolog structures to do that, am I right? ... 阅读全帖 |
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f******g
发帖数: 1003 | 35 http://www.merckmanuals.com/vet/pharmacology/anti-inflammatory_
&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&
Biochemical mediators released during inflammation intensify and propagate
the inflammatory response (see Anti-Inflammatory Agents: Actions of
Inflammatory MediatorsTables). These mediators are soluble, diffusible
molecules that can act locally and systemically. Mediators derived from
plasma include complement and complement-derived peptides and kinins.
Released via the classic or alte... 阅读全帖 |
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b******n
发帖数: 4225 | 36 嗯，一般DnaK和DnaJ是一起的，用ATP/Mg++也许见效
实在不行试试denatured soluble protein或者glycerol等偏方
而GroEL用ATP/Mg++比较好解决 |
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s*********y
发帖数: 387 | 37 thanks. no DMSO is used in our system.
All in PBS or water buffer. the solubility of the peptide is very good.
Also, the Biotin-conjugated IgG has the same phenotype, but we can see an
obvious bell shaped curve, for the peptide it is a exponential growth curve,
making the interpretion intriguing... |
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w*****s
发帖数: 230 | 38 附件是主治大夫的reasoning(email回复，红字)， 他的水平我们还是很佩服的，因为
他已开始猜了个病就和我们宝宝症状非常一致，可惜后来发现不是；而且他不是代谢病
专家。
问题是坚持宝宝不吃长链脂肪酸的奶粉后，他奇迹般地改善了；完全出乎医生们的预料
。我感觉医生列的是常见的代谢病，而我们感觉宝宝吃长链脂肪酸似乎是慢性中毒，大
概需要一个月左右才能体现出来。因为过程很慢，我们怀疑很多方法查不出来。
我们最大的问题是如果是这种病怎么查？不能确认的话他一辈子吃饭都受影响，基本也
就很难出门时间长一点了。尤其是他的肝已经在要换的边缘。医生们现在改口说他的病
程都是偶然，可我们不能同意。为什么每次都是长链吃一段时间就得病，尤其是这次停
了鸡蛋后马上改善。。
谢谢大家。 （下面是文字，可惜设置不成红色）
Q: Our biggest concern is the reason of his liver damage.
A: The biopsy shows severe fibrotic changes and tells us that this is not a
liver that ... 阅读全帖 |
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S******e
发帖数: 393 | 39 将S1 (cytosolic protein) 和 S3 (nuclear soluble protein?) 去掉后，剩下的
pellet再用0.1%的Triton extract, 收集到的上清暂称为X，想知道X是些什么蛋白？
Chromatin associated protein?
方法如下：
1. Cells were extracted with Buffer A (10mM Hepes pH7.9, 10mM KCl, 1.5mM
MgCl2, 0.34mM Sucrose, 10% Glycerol, 1mM DTT plus protease inhibitors)
containing 0.1% Triton for 5 min on ice, centrifuge 1300g X5 min, discard
supernatant (S1), wash once with BufferA and discard supernatant.
2. Extract cell pellet with Buffer B (3mM EDTA, 0.2mM EGTA, 1mM... 阅读全帖 |
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h***a
发帖数: 145 | 40 Soluble adhesion molecules as markers for sepsis and the potential
pathophysiological discrepancy in neonates, children and adults
Rens Zonneveld , Roberta Martinelli , Nathan I Shapiro , Taco W Kuijpers ,
Frans B Plötz and Christopher V Carman
Thank you in advance. |
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l****5
发帖数: 2968 | 41 下面很多papers，没有学校账户查不到了。请大家帮忙查一下吧。请发greenfast2013@
gmail.com
包子不多，不过由于文章很多，所以会发包子以表感谢的。万分感谢啊！
1) Effects of high electric field pulses on enzymes
2) Neuropharmacological investigations on muscimol, a psychotropic drug
extracted from Amanita muscaria
3) Extraction of BaChu mushroom polysaccharides and preparation of a
compound beverage
4) The effect of the high pressure homogenisation on the activity and
stability of a commercial neutral protease from Bacillus subtilis
5) Isolation o... 阅读全帖 |
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s******s
发帖数: 1584 | 42 我觉得用SDS buffer 煮一下可以增加 solubility 很多膜蛋白都不溶的 |
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x****s
发帖数: 29 | 43 J Neurovirol. 2014 Jun;20(3):269-77. doi: 10.1007/s13365-014-0244-8. Epub
2014 Mar 27.
Pentosan polysulfate treatment ameliorates motor function with increased
serum soluble vascular cell adhesion molecule-1 in HTLV-1-associated
neurologic disease.
http://www.ncbi.nlm.nih.gov/pubmed/24671717
非常感谢。 |
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m*********D
发帖数: 1727 | 44 Water soluble--you can find the information with the compound, up to 50 mg/
ml.
However, we generally make 10 mg/ml.
We keep water solution at -20. For how long? I have no idea.:(. At least a
few months should be ok. |
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m*********D
发帖数: 1727 | 45 Water soluble--you can find the information with the compound, up to 50 mg/
ml.
However, we generally make 10 mg/ml.
We keep water solution at -20. For how long? I have no idea.:(. At least a
few months should be ok. |
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M********a
发帖数: 35 | 46 大部分的TF binding site都是在nucleosome free region,也就是DnaseI HS sites，
但是如果chromatin没有solublize好，因为crosslink和sonication的bias,会导致
input也出现DnaseI HS的pattern,甚至有一种方法就是用这个原理去identify HS
sites的。一般ChIP protocol用150mM的盐，但是最好是300mM,盐浓度太低会导致
chromatin溶解不了。 |
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o*****4
发帖数: 1245 | 47 水溶蛋白也不好结晶.结构稳定的不管膜蛋白水溶蛋白,都好结晶.结构不稳定的连
soluble protein都拿不到，何谈结晶。
好多难的膜蛋白并不是结不出，而是根本纯化不出，水溶蛋白也一个样。
单论结晶这一步，膜蛋白是tricky一点。
的。 |
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D***H
发帖数: 30 | 48 4.7A 的晶体也能发Science，2013年的
Crystal Structure of a Soluble Cleaved HIV-1 Envelope Trimer
：其实做结构挺心酸啊，生物其他分支的人都说做结构的：哟好发文章啊，还发CNS。
。。
： |
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Z**********g
发帖数: 222 | 49 本人博士期间是做癌基因信号通路的，打算换个方向做postdoc。感觉最近肿瘤免疫大热
，工业界的机会也很多，想跳啊。想问问牛人们具体哪些方向最promising：
T细胞治疗方面（本人最感兴趣）：
1） CAR-T 细胞治疗：火了很久，但成功的貌似只有针对CD19的B细胞血液肿瘤。而且
只能针对细胞表面抗原。
2）Natural TCR engineering：鉴定出与肿瘤相关抗原-MHC复合物高亲和力的TCR
，然后engineered到T细胞。似乎可以靶向大多数的细胞内抗原？
3）Soluble biologics：基于抗体或TCR的生物分子，注射到体内，激活T细胞。
ImmTACs就是干这个的。
4）Immune checkpoint：目前极为火热，包括CTLA4和PD1抗体。
B细胞治疗方面：
通过肿瘤抗原免疫而产生抗肿瘤抗体；貌似这方面木有什么进展？
请问领域的大神，该跳哪个？ |
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w*********o
发帖数: 3030 | 50 I am not sure. It might still doable unless your current funding is through
this mechanism already. 14-279 covers:
"In vitro cellular and tissue activities (potency, selectivity, specificity,
etc.);
In vitro structural, physicochemical, and biochemical properties (solubility
, stability, membrane permeability, protein binding, microsome stability,
metabolite identification, CYP inhibition, etc.);
In vivo pharmacokinetics (PK) with absorption, distribution, metabolism,
excretion (ADME) and toxici... 阅读全帖 |
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