b******y
发帖数: 627 | 1 It will be expensive from Anaspec. should try luck with smaller company for
this.
Do you have friends who can help you with this? It is not different from
normal peptide synthesis except that the protection on the two -NH2 on the
first Lysines are the same probably fmoc. You also don't mind a mixture.
There is one more strategy if your peptide doesn't have cys in it.
Synthesize your peptide with an extra cys at the C-terminus. Buy tri-
maleimide from somewhere. Mix your peptide with tri-maleimid... 阅读全帖 |
|
|
b******y
发帖数: 627 | 3 Not complicated for the last strategy. free thiol from cys reacts with
maleimide very efficiently. tri-maleimide is cheap. you have to synthesize
peptide anyway.
Just a reminder: the N-termini of proteins in vivo (bacteria or mammalian
cell) are complicated. The majority of them have the met cleaved upon
translation. Also the majority of them are N-terminally acetylated
cotranslationally or post-translationally.
Be careful! Just want to remind you not to use wrong peptides for Ab
generation. |
|
b******y
发帖数: 627 | 4 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 |
|
s******y
发帖数: 28562 | 5 不好意思，得问一个白痴问题，在C-terminus 加的那个cysteine,
是要C-terminal free acid 形式呢，还是 amide 形式？就是末端那个羧基
的处理是否有讲究？
另外，能不能麻烦你给我贴个连接或者直接和我说说用tri-maleimide
合成dendrimer时候的反应条件？Buffer salt, pH, DTT?, temperature,
time, termination of reaction，等等。
for
e. |
|
n******g
发帖数: 662 | 6 I did follow Pierce instructions for COOH to amine-protein link in water
solution using EDC/NHS activation or just EDC activation.
Its protocol did work well for polymer microbead-COOH and amine-protein
linking. But it seems not working if I change microbead-COOH to micelle-COOH.
I do see people using maleimide-PEG-Phosphilipid to react with thiolated
protein.
without |
|
c*****u
发帖数: 439 | 7 嗯 感谢这么详细的答复
www.
maleimide |
|
s*******g
发帖数: 36 | 8 想把protein immobilized到sensor chip上，用biacore或者octet Red做screen。。。
可是问题是我label的蛋白一直load不上去，load到的信号非常低。。。。 我用了
maleimide-PEG4-biotin 去label cysteine，结果一直不好。信号就是很低的时候就达
到平衡了，很奇怪。 之后我换成NHS-LS-biotin，这个大家用的比较多，结果load的信
号更低。。 我都有BSA去test labeling，都信号很低。。
我现在的问题是，蛋白肯定是label了，30%左右，我用的1 biotin： 1 protein的
ratio做的labeling，因为不想over label multiple sites on one protein. 都做了
两个月了，快绝望了。。。什么条件都试了，Octet red的信号就是不行！
另外，有人说BSA 不好label，想问问别人有用其他蛋白label的吗，我想做个positive
control， 说明是蛋白的问题，还是label的方法的问题
有经验的人指点一下吧。谢谢了！ |
|
n********e
发帖数: 1630 | 9 多谢。终于找到明白人了。我dialysis没有那么多 。我用500x 1h Twice. 这个可能真
的不够。可能有free biotin在 。 下次我一定试试你的dialysis的方法。我octet 信
号才3-4 nm 有的时候更低。就是很低就饱和了。确实说明binding site没了。
另外我是label 4°C 过夜。这样就成了三天时间label一次了。。。
另外我用的是maleimide-PEG2-BIOTIN 50mM HEPES buffer. 标记SH group
另外我也有NHS -LC-BIOTIN... 本来想用BSA 去label做个control 结果bsa也load不
上去
听了你说的。我感觉真的要用更多的dialysis
待会私信再问你。谢谢啊。。。
hours |
|
l**y
发帖数: 595 | 10 反应物在水利溶解度不是很好，可以加点甲醇或者DMSO吗 |
|
t**w
发帖数: 82 | 11 methanol is ok.
DCM, THF, toluene works too. |
|
|
s****2
发帖数: 4569 | 13 if pure 有机溶剂, add tertial base (2eq TEA or DIEA). If in aq. solution,
can use DMF or DMSO to help to dissolve,pH 7-8 bah. |
|
p****y
发帖数: 50 | 14 为什么跟硫醇而不跟醇或胺加成反应？选择性很强， 什么原理？
恳请赐教！ |
|
f***o
发帖数: 161 | 15 S is more nucleophilic than the other two. |
|
j***i
发帖数: 61 | 16 maleimide coupling
接在Cysteine上面 |
|
l****e
发帖数: 157 | 17 最简单的办法就是给你的小分子连一个MALEIMIDE,然后和CYSTEINE反应.也可以连NHS,
然后和LYSINE反应,不过一般会有很多LYSINE. 最后,如果你想特异地连在某个位置,而
蛋白中CYSTEINE也有多个的话,你可以用SCHULTZ的办法在你想的位置引入一个KETONE,
然后和HYDROXYLAMINE或HYDRAZINE DERIVATIVES反应.当然你也可以引入一个AZIDE或
ALKYNE,然后做CLICK CHEMISTRY,但这个在蛋白上做比较难. |
|
|
|
l**y
发帖数: 595 | 20 需要一个maleimide end functionalized polystyrene sulfonate
大侠们知道哪个公司可以吗 |
|
c*s
发帖数: 2145 | 21 A
ABA
acrylonitrile-butadiene acrylate
ABS
acrylonitrile-butadiene styrene terpolymer
ACS
acrylonitrile-chlorinated polyethylene styrene terpolymer
AMA
acrylate maleic anhydride terpolymer
AMMA
acrylonitrile-methyl methacrylate
APO
amorphous polyolefin
ASA
acrylonitrile styrene acrylate
B
BMC
bulk molding compound
BMI
bis maleimide
C
CA
cellulose acetate
CAB
cellu |
|
m*****e
发帖数: 1506 | 22 来自主题: Macromolecules版 - 外行求助 except Maleic anhydride and Maleimides |
|
k******e
发帖数: 32 | 23 问题弱点，莫笑！
想要的一个前端共聚物目前没啥好的制备方法，也买不到。
聚合物A和B上分别有能相互反应的端基官能团。把它两混合一块，能有多大的反应几率
呢？比如A的分子量为10k, B的分子量为5k. 一个带巯基，一个带Maleimide。反应后如
何把嵌段共聚物提取出来呢？？ |
|
