a*****a
发帖数: 640 | 1 我刚才第一反应是你要测抗体
刚才google一下
The technique used to determine the coupling efficiency of peptide to BSA
depends on how the peptide was conjugated. The common method of coupling
through terminal cysteines makes coupling efficiancy determination simple.
Just
do an Ellman's assay (dtnb).
Coating a peptide on an ELISA plate has a few inherent problems. To begin
with,
the interaction between plain polystyrene plates and small peptides can
often
cause degradation of the peptide. You may want to try a diffe... 阅读全帖 |
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c***I
发帖数: 238 | 2 【 以下文字转载自 Rite_aid 俱乐部 】
发信人: catII (little+cat), 信区: Rite_aid
标 题: 请问有人听说过心脑康肽茶(health heart + brain peptide)
发信站: BBS 未名空间站 (Sun Jan 13 12:45:54 2013, 美东)
国内亲戚让帮着打听一下。搜了半天health heart + brain peptide根本找不到这个东
西，据说是美国产的，美国FDA apprvoed。
唯一能找到的相关英文网站就是这个：http://us-peptide.com，而且没有contact info。
试着查了一下FDA approval，可能是我太弱了，根本查不到，另外搜索Dr. Hongguang
Cheng也找不到任何文献，虽然http://us-peptide.com/about.html claim他had been invited by Harvard University for academic exchange in 2004, and he joins
the development t... 阅读全帖 |
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l**********1
发帖数: 5204 | 3 利用人工智能免疫系 likes CD44 plus Hyaluronan (hyaluronic acid, HA) is a
linear naturally occurring polysaccharide 钓3kDa左右的多肽的鱼
可以不通过分离而直接挑出peptide or protein来特异反应--just matrix two cents.
References:
1)
Erickson M, Stern R.
Chain gangs: new aspects of hyaluronan metabolism.
Biochem Res Int.2012:893947.
//www.ncbi.nlm.nih.gov/pubmed/22216413
and
2)
Chaim OM et al.(2011)
Brown Spider (Loxosceles genus) Venom Toxins: Tools for Biological Purposes.
Toxins (Basel). 3: 309-344
Abstract
Venomous animals use ... 阅读全帖 |
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w******e
发帖数: 1187 | 4 多谢建议！
是这个。说copy number是每个beads上要coat不同的peptide。btw有什么好方法
测peptide浓度吗？如果已知peptide的部分序列的话
这个倒不知——ab biotinylation应该有满成熟的protocol吧？问题是我希望
每个beads（1到几um直径）上coat至少1e4 peptide,最好1e5以上。。。
要做一个peptide library的话，有点担心用GST做constant tag会不会太大？
有没有可能modification-by-synthesis的方法，加入一个biotin-AA analog，只在
某个位置incorporate的？继续伤脑筋ing |
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b****y
发帖数: 81 | 5 no. Just wondering whether there is any sucessful example of a antibody that
can recognize a phos-peptide.
Most antibody is generated using peptide, in your case, it is probably
generated from phos-peptide. Are you detecting protein or just peptide? If
just peptide, using mass-spec is probably easier. |
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A******y
发帖数: 2041 | 6 A lot of antibodies for phos-"whatever protein" is generated from phos-
peptide as antigen, and the resulting antibody only recognize the phos-
peptide on the protein. It will also recognize the phos-peptide; therefore,
you can block the antibody binding using the peptide as competitive control
. You probably can do an ELISA if you want to see if the phos-peptide is
there or not. Btw, there are antibodies can even recognize a single
phosphorylated amino acid. Just go to any commercial antibo... 阅读全帖 |
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b******y
发帖数: 627 | 7 Labeling a short peptide is not hard at all, especially so if you are
ordering the peptide from a company. They can do a variety of fluorophore
labeling.
If you are going to DIY, as suggested by LS, try Cys or Lys or N-terminus
labeling. For example, if there is single Cys or affordable to add one, you
can buy different Maleimide-fluorophore and do the conjugation yourself. If
there is not Cys available, you can buy NHS labeled fluorophore and do the
conjugtion with the N-terminus and/or Lys.
Pe... 阅读全帖 |
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C******T
发帖数: 300 | 8 LCMS 和HPLC 都只能大致判断peptide的纯度，无法定量。
这两种方法（LC和HPLC）是依靠紫外峰估算peptide的含量。很多时候样品里的salt
and small molecule impurity不能依靠紫外吸收分析。业界认可的定量方法是楼上说
的AAA analysis。所谓 75%， 90% peptide content, 指的是AAA analysis 得到的数
据。
一个peptide经过HPLC提纯，看似纯度达到95%，做一下AAA analysis，发现peptide
content 只有50%，是很常见的事情。
不过楼上的样品，根据MS谱图来看，没有做AAA analysis的价值，太不纯了。50AA左右
的多肽，应该只有四五个峰。机器灵敏度不高的话，一般就是两三个峰。 |
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h*****t
发帖数: 1226 | 9 only one peptide? Not possible.
If you have several peptides matched to a protein, you may use
peptide finger print to assign the possible peptides. |
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A******y
发帖数: 2041 | 10 Most antibody is generated using peptide, in your case, it is probably
generated from phos-peptide. Are you detecting protein or just peptide? If
just peptide, using mass-spec is probably easier. |
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e****e
发帖数: 1042 | 11 According to your opinion, I should use Bradford assay to determine total
protein and peptide. I will get the total amount of my peptide product from
HPLC. Then the protein amount is total protein and peptide minus the amount
of my peptide product. Dialysis is not necessary, right? |
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e****s
发帖数: 1125 | 12 其实艳阳天说的方法很好。
你就测总蛋白，Braford之类。然后通过Gel filtration分离Peptide一定量总蛋白里面
的量。比如上10mg的蛋白，然后回收Peptide峰，通过A280算出Peptide量。这样
Peptide在总蛋白中占多少比例不是很清楚了？
精确测蛋白浓度是很麻烦的，一般都是相对浓度。不论是Braford还是A280都存在不同
蛋白间的差异。
is |
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b******y
发帖数: 627 | 13 If you have a 50 a.a peptide with no particular 2nd structure, I call it a
peptide. Some polypeptides, like ww domains, have only about 40 a.a. (3
short beta strands packed in an antiparallel fashion), which can bind
proline-rich peptide ligands. I can call them proteins or at least protein
domains. I believe there are a few domains that are of comparable size or
even smaller than ww domains. Don't remember what they are called on top of
my head.
As we all know, it is so trivial to translate DNA... 阅读全帖 |
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c******y
发帖数: 193 | 14 最近在做peptide drug,在做in vivo动物实验时发现peptide非常容易被酶解。
请教版上牛人有没有相关文献或者书籍推荐来改造peptide的结构，实现long
circulation peptide drug？
非常感谢！ |
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c******y
发帖数: 193 | 15 Thank you all for the reply!
1) Which amino acid in the peptide should be changed to d-AA? or any amino
acid change could prevent the peptide degradation.
2) Cyclization means make the whole peptide be cyclic, or just part of it?
Are there any reference on the cyclization protect peptide?
3) thanks for sharing peptoid! Are there any review on this?
Thank you again! Good night, sleep tight! |
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p******n
发帖数: 2 | 16 Employer: American Peptide Company (APC) http://www.americanpeptide.com/
Location: Sunnyvale, CA, 94086
Position: Peptide Chemist(current opening, even not show on our website)
Requirement:
1) Master, or Ph.D in organic/analytic chemistry;
2) With experience in peptide synthesis/purification is a plus;
3) Highly motivated individual;
4) Good oral and written communication skills;
Contact: [email protected] or L***[email protected] |
|
o****e
发帖数: 1011 | 17
把这个interacting peptide的一端延长出去接个biotin；（尽量选择“不影响该
peptide和你要测的蛋白之间的相互作用”的一端）
选择使用Streptavidin （或NeutrAvidin） Coated的Plate，你的biotinylated
peptide应该能接到其表面上。 |
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w******e
发帖数: 1187 | 18 请教：用cell-free expression synthesize peptide/protein, 要求newly synthe-
sized peptide/protein自动被immobilize到beads上，并达到尽量高的copy number。
最直接的方法大概是在gene里加个FLAG/HA的sequence，beads上coat FLAG/HA
antibody。但Ab个头太大担心copy number上不去。
请教有什么alternative method？比如his6-peptide的coverage会不会更高？
比如有没有site-specific biotinylation的reagent可以跟cell-free expression
合用？
非常感谢！ |
|
s******y
发帖数: 28562 | 19
如果你们有相对应的抗体的话可以简单的用免疫荧光法来测定
的确会有些大，而且会影响你的peptide comformation
有某些特定的氨基酸可以，比方说lysine （侧链被biotinlylated) 我忘记是
哪个公司有生产的了。你可以去骨狗一下。
不过，事先得警告一下，这个肯定会影响你的peptide构型。除非你故意把lysine
放在所有peptide 最后一位。 |
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y*****w
发帖数: 146 | 20 请问大家，有谁知道什么peptide在ER里聚集会导致apoptosis？现在做的project需要
用到一个可以在ER内导致cell apoptosis的peptide， 本人对ER的了解很少，看了一些
review（估计没找对）也没有头绪，谢谢大家指点一下。当然peptide是最理想的，实
在没有small protein也可以。谢谢了！ |
|
e****e
发帖数: 1042 | 21 I already got what I need to know. My question is very simple. That is, is
there any method can measure only protein but no peptide without further
seperation or purification? I thought the answer should be "No" before I
came here. But I am not so sure since I never think about it before. When I
worked in purification labs, we always used OD280 to determine protein
concentration since the protein is quite pure. When I worked in molecular
biology lab, we always used Bradford to determine the pro... 阅读全帖 |
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C******T
发帖数: 300 | 22 并不是所有公司会给出peptide content。据我的经验，纯度数据会免费提供。大部分
公司peptide content会额外收费，一般100左右，贵的可能会200到300。我同意你的说
法，LCMS可以给杂质定性。有两点我觉得可以商榷，第一，透析能不能去除盐。我们一
般先desalt, 然后HPLC提纯多肽（～95% pure)，然后做peptide content(假设70%)。
这25%的difference一般是多肽上结合的盐，比如TFA。众所周知多肽是以盐的结合体形
式提纯出来的。透析是不必要也是无益的。第二，多肽的不纯物当然会影响到结果。关
于小分子对生物学有没有影响，别的杂质不说，就举一个简单的TFA。业界的animal
study protocol，一般会要求避免多肽以TFA盐的形式进入动物体内，首选是acetate
salt，病理学毒性比TFA小 up to 30%。其他的小分子就更不用提了。TFA影响生物学的
例子我自己也见过一些。 |
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v**********t
发帖数: 9 | 23 感谢专家们的建议！
这个peptide确实比较长，60aa。因为是要用于structural study,所以比较关心全长
peptide在peptide总量中的百分比。盐的话可以替换。 |
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k****o
发帖数: 728 | 24 Considering peptide is too small, is SDS-PAGE capable of separating 3000 Da
peptide from 6000 Da peptide ?
want
to |
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s*****d
发帖数: 110 | 25 小弟最近在做个实验， 希望能有尽量高的peptide mapping coverage， 但是有几个
peptide做了几遍都看不见， 有什么方法或者调什么parameter可以把它搞出来啊？
我用的是trypsin digestion， dionex 3000+ LTQ
希望大家帮帮忙啊 多谢！！
update: 是个recombinant peptide (117 amino acids) |
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l***d
发帖数: 1828 | 26 【 以下文字转载自 Biology 讨论区 】
发信人: lrpwd (lrpwd), 信区: Biology
标 题: 有什么算peptide分子量的工具
发信站: BBS 未名空间站 (Tue Jun 19 10:01:38 2012, 美东)
用MALDI测小peptide,MS/MS分析碎片,请问有什么算peptide分子量的工具和网站吗?想
计算碎片,谢谢 |
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q*****8
发帖数: 23 | 27 诚心请教,有关peptide/protein 药的几个问题
1。原理？peptide/protein药不会被酶水解掉么?
2. 制药公司做这方面的多吗？
3。peptide/protein药前景如何？
谢谢了！！！！！！！！ |
|
h*****t
发帖数: 1226 | 28 Yes. Those information will definitely help to optimze the experiment
conditions.
How big is that peptide mssing on column? sometime big peptide would just
be too hydrophobic, change to C8, C4 column for separation would help.
And I think he was using a Dionex Ultimate 3000 with trpaping column,
whether the peptide can be efficiently trapped is also a question.
c18 column) |
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I****7
发帖数: 6 | 29 Position Overview
LifeTein, LLC is seeking a technician to join our team of dedicated
professionals focused on peptide synthesis. The ideal candidate will be
motivated, energetic, forward thinking, and possess outstanding technical
skills, organizational awareness, and a commitment to excellence.
Major Responsibilities will include, but are not limited to the following:
Characterization and identification of compounds using modern analytical
methods including, but not limited to, HPLC, LCMS, and... 阅读全帖 |
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G*****j
发帖数: 157 | 30 前几天皮肤觉得很dull，然后我一个朋友推荐我用了skin biology 的copper peptide
产品，我自己给自己配了CP Serum and Exfol serum.
CP serum 含有copper peptide, 中文所谓的蓝铜胜钛和晚上用 Exfol serum，然后两
者之后都用 La Mer.
一周下来皮肤光泽很多！
http://baike.baidu.com/view/2900629.htm ->蓝铜科普~ |
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A***l
发帖数: 302 | 31 4. Multiple antigen peptide (MAP) approach
In order to avoid possible undesirable side effects of adjuvants/carriers,
another approach, MAP has been applied to the development of synthetic
vaccines. The goal of it is to design chemically unambiguous, multivalent
vaccines containing the relevant B- and T-cell epitopes but requiring no
adjuvants/carriers. This approach uses a small peptidyl core matrix bearing
radially branching synthetic peptides as dendritic arms. The simplest
design is to util |
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l*****k
发帖数: 587 | 32 because of the nature of my work, I just hear a really exciting news. It
is about drug design that produced peptide with almost identical activity
as the cell surface's natural conjugates(an import hormone). Sorry, I don't
think it is appropriate to reveal more details about it.. The peptide is
much, much easier to produce than the real hormone.
I wonder if we can talk about the prospect of this kind of research, I
personally think it is a revolutionary success, it changed the whole way we
can t |
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M****e
发帖数: 70 | 33 talking about de novo peptide-derived drug design, i have an
idea using biosensor for such investigation. basically, i
would like to assess and evaluate the binding affinity of the
peptide to the known receptor as well as its downstream signaling
transductivity. so i may design two chips (biomembrane-based),
both approapriately covered by the receptors, with one linking
to some biosensor that basically detect the affinity, while the
other coupled to the primary downstream signal transducer while |
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r****o
发帖数: 105 | 34 I have similar experiences as yours. That is about determination
the anomericity of glycosylation on EGF repeats. It involves
processing the small domain EGF into peptides, and then subject them
to glucosidase digestion.
I would suggest you using HPLC with either Reverse Phase or Gel filtration
column(like superdex from Pharamacia). This is the best way to recover
small amount of peptides. If your sample has quite a lot and the MW is
big enough, you can try acetone precitation to get rid of |
|
m**z
发帖数: 787 | 35 For your sample, 0.2uM filter will be fine. Don't know what kind of
containers you are using... But I still don't think it is that important...
I don't have much experience with small peptides.For proteins, buffer with
salt will usually be better than just water. Adding some detergent may also
help if you don't think it will interfere with your experiment. Also, note
that there may be TFA remaining in the lyophilized powder. So just adding
water will result in an acidic solution.
The only way I ... 阅读全帖 |
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K******S
发帖数: 10109 | 36 don't worry, peptides are VERY stable. especially your peptide, I can't
think of any proteolytic enzyme that can cleave the sequence.
If you can, just make some aliquots |
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c*****e
发帖数: 436 | 37 it seems the Chemist synthesized this peptide told me that in this peptide
valine or some thing? can be oxidized? (don't know what does this mean).
And I saw from papers other people make similar sample with very complicated
steps with:
"Samples were prepared for the NMR experiments in 6-mm tubes by dissolving 0
.3g of lyophilized material in 1.5ml of distilled H2O. ..The tubes were
placed in a bath at 20C, and argon gas was passed through the samples for 6h
by means of long-tipped pipets placed... 阅读全帖 |
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m**z
发帖数: 787 | 38 The Val in your peptide won't get oxidized... Argon is for anaerobic purpose
. But since your peptide doesn't have Cys, don't think it is necessary. Don'
t know about your question 2... They didn't sterile as what I can tell from
their description.
For NMR, it should be easy to check your sample. Run a simple HSQC at the
end of your experiment to compare with the spectrum taken at the beginning.
This should give you a pretty good idea of your sample integrity.A little
precipitation during NMR ma... 阅读全帖 |
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w******e
发帖数: 1187 | 39
sorry, 没看懂-_-
我的目的是测一个蛋白，想用该蛋白的一个interacting peptide做capturing agent，
用aptamer做detection agent，用peptide instead of Ab做capturing agent的
原因是1. 担心aptamer跟ab有steric hindrance；2. 不想用ab呵呵 |
|
o****e
发帖数: 1011 | 40 EDC chemistry本身没什么，尤其人家把kit/protocol都弄好了话。
主要问题是比如你的interacting peptide上的那些COOH和该plate上的那些NH2（脱水）反
应后，其和你要测的蛋白之间的相互作用恐怕就已经和“原始的interacting peptide和
你要测的蛋白之间的相互作用”有很大差别了。。。 |
|
s******y
发帖数: 424 | 41 我没有什么molecular biology 的背景，希望想请教一下。
我通过mass spec可以sequence出来novel peptide，但我怎么查相应的gene里能不能
encode出来这段peptide呢？是不是应该用ncbi的blast protein，但查出来的number 好像是protein 的啊？blast neucleotide查出来的是不是就是gene的accession #了啊 |
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b******y
发帖数: 627 | 42 Labeling via Cys and Lys suffer the same problem as ordering from company
because the fluorophores available will be the same as those provided by
peptide synthesis company.
One idea though, can you 1) biotinylate your peptide, 2) deliver to the cell
, and 3) fix and then IF based on SA? |
|
y******8
发帖数: 1764 | 43 Radioactive labeled peptide is the easiest, then.
Endogenous biotin is also problematic. On the other hand, biotin may not be
small enough for your peptide. |
|
e****e
发帖数: 1042 | 44 现在需要把yeast extract里面的protein含量测出来。我们要的是其中的peptide。但
是想知道里面protein的杂质的量。请问什么assay可以测出来？Bradford protein
assay和Bio-rad protein assay 应该是区分不出peptide和protein的吧？谢谢指教！ |
|
e****e
发帖数: 1042 | 45 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 | 46 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 |
|
e****e
发帖数: 1042 | 47 As I said before, I've worked on protein projects for a long time. But I
never think too much. Once a person who never works on this field ask such
an question, I lost for a moment. Just like when a kid saw a piece of white
paper turns to black when the paper was burned, he asked why the white turns
to the black. I told the kid the fire make the paper to carbon. Then the
kid said: "So the paper is not carbon?" I suddenly realized the paper is
also made of wood and it should be carbon. Then I nee... 阅读全帖 |
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b******y
发帖数: 627 | 48 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. |
|
b******y
发帖数: 627 | 49 Finally to LZ, you can quantify your product using HPLC at any stage of your
purification. Meanwhile measuring total protein including protein, peptide
and your product using branford or methods alike, you will know the
enrichment or purity at any stage. I believe this is more or less how your
company is doing it.
What makes me agitated is:
Even if you can magically distinguish 3 KDa peptides from 1-2.9 KDa and 3.1
KDa-infinite, you still don't know whether they are purely from your product
or p... 阅读全帖 |
|
l***d
发帖数: 1828 | 50 用MALDI测小peptide,MS/MS分析碎片,请问有什么算peptide分子量的工具和网站吗?想
计算碎片,谢谢 |
|
