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全部话题 - 话题: crosslink
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d****7
发帖数: 109
1
书里都是那么说的,具体细节我估计没人清楚。而且formaldehyde crosslink protein
-dna只是理论上的,如果你看最早的那篇发明ChIP的文章,文章里说,在in vitro的情
况下,formaldehyde只能有效的crosslink nuceosome-dna interaction,但是却不能
crosslink TF-DNA (他们似乎只测试了lacz)。
另外,modENCODE的Julie Ahringer跟我们说,她和Steven Henikoff都觉得
formaldehyde不能有效的crosslink protein-dna,而大家在做ChIP的时候,只是将
nucleosome crosslink到DNA上,然后其他的TF只是crosslink到nuclesome上了,间接
的crosslink到DNA上。
对于reverse crosslink,只是习惯性的加热overnight。
formaldehyde的浓度,大家用的差别还是挺大的,有些人用1%,有些人用1.5%,有些人
室温crosslink,有些人37度,有些人在formalde... 阅读全帖
s*********i
发帖数: 53
2
刚开始学ChIP,查了很多资料也还没搞明白formaldehyde crosslink究竟是怎么回事。很多比较老的书里边说是形成了-CH2-键,或者是-NH-CH2-键。但是如果是这两个键,那65度5小时的reverse crosslink是不太可能的吧?
另在某些论坛上看到有人说是形成了amid bonds, 这样就是加热可逆的。可以理解。(但是reference????)
另外,Ren et al.2000年的Science paper 并没有写有reverse crosslink, 难道是后来人家补充上去的?ChIP时crosslink的程度和reverse的情况还是蛮关键的吧。所以想弄清楚怎么回事,然后选择formaldehyde的浓度和fix的时间。
大家帮忙看看!!多谢~~~
z*******a
发帖数: 175
3
来自主题: Biology版 - help: crosslink co-immunoprecipitation
I have been trying to show protein interaction between a kinase and its
predicted substrate by regular co-
immunoprecipitation. but failed twice already.
boss suggested crosslink co-IP again and again, very nicely though. I heard
it is almost impossible to publish
protein interaction data coming out from crosslink co-IP.
every Daxia,
Anybody see any published paper having crosslink co-IP data in it? If you do
, please forward me the basic info
( journal and author or title). Thanks a lot.
any ot
m******i
发帖数: 73
4
check this paper.
http://www.ncbi.nlm.nih.gov/pubmed/20178313
the -CH2- is weak for the crosslink induced by formaldehyde.

。很多比较老的书里边说是形成了-CH2-键,或者是-NH-CH2-键。但是如果是这两个键
,那65度5小时的reverse crosslink是不太可能的吧?
(但是reference????)
后来人家补充上去的?ChIP时crosslink的程度和reverse的情况还是蛮关键的吧。所以
想弄清楚怎么回事,然后选择formaldehyde的浓度和fix的时间。
f*****f
发帖数: 195
5
我自己一般用DMP 自己crosslink,没用过那个kit。
crosslink也没什么繁琐步骤,kit和买粉末应该也没啥区别。
一般magnetic beads总体上要贵些,好处是可以用磁铁吸附,大小很均一,而且不像
agarose那样容易变形,不必离心。

Crosslink
magnetic
m*********r
发帖数: 49
6
都准备从Sigma买Protein A beads了然后自己crosslink了,又从Thermo搜到Crosslink
+ CoIP的kit,用的是disuccinmidyl suberate (DSS),貌似也不贵多少,还省事。有
人用过这个的推荐么?Kit好像也有magnetic和agarose的可以选,是不是一般magnetic
的方便点?
g*********3
发帖数: 177
7
It seems you have trouble in reverse-crosslinking step. show us your recipe
for reverse crosslinking. Run a gel on dna.
d****7
发帖数: 109
8
我刚做ChIP的时候也遇到过这种情况,这种现象可能是factor specific
这样吧,你在跑ChIP的时候,再多跑一下两个样品:
(1) un-crosslinked whole cell extract
(2) crosslinked but un-sonicated chromatin
如果(1)都没有目的条带,证明抗体根本不识别蛋白,或者蛋白根本不表达。但是既然
你说是knockin tag,也许表达亮太低
如果(1)有带,(2)没有,说明蛋白的epitope可能被formadehyde毁掉了
如果(1) (2)都有带,超声后的input没有带,证明oversonication,把蛋白给毁了,我
最近就遇到这种情况,都是input,超声前能检测到带,超声后就没了。。。。
还有,煮10分钟足以,30分钟有点过
U**s
发帖数: 3390
9
the cell density is too high. you may try the formaldehyde crosslink in 10ml
growth medium (w/o serum; 1% formaldehyde) in falcon tubes.
If you grow adherent cells in monolayers, it's better to perform the
crosslink in petri dishes.
The basic principle is that: you want to take a snapshot of all kinds of
interactions with the minimum disturbance to the cells.
e****d
发帖数: 16
10
I am a new member here. And have a problem to ask for help.
我合成了一种聚合物, 合成过程中出现了爬杆包轴的现象,不溶。如何表征以证明发生
了交联?只有DSC一种方法么? 谢谢。
I have synthesized an unsoluble polymer. During the polyerization, it showed
to be crosslinked. How can I make charaterization of it to confirm it has been
crosslinked? Is DSC the unique way?
If I change the reaction conditions, a soluble polymer can be obtained.
Thanks.
w********h
发帖数: 12367
11
you can use this as a rough estimation.
but some materials just have crosslink-like behavior, not fully crosslinked.
w******r
发帖数: 43
12
来自主题: Macromolecules版 - chemical or physical crosslinking?
1. What kind of filter did you use, nylon type or Teflon type? Or did you try
didfferent type filter?
2.You can try to dissolve your polymer in DCM and do the filtration again.
I do not think it was crosslinked. Even if there are some crosslinks in your
polymer, you should still be able to see some signals in the GPC trace.

blah)
membrane
can
j*******8
发帖数: 933
13
来自主题: Biology版 - 怎么做crosslinking?
亲爱的同行们,俺现在第一次试CROSSLINKING, 上礼拜跑了一次, 根本就进不到胶里
(4-15%的胶)。 请问做过这个的同学们, 用什么电压跑胶, 什么电压转膜? 拜谢
了!
W****C
发帖数: 1937
14
来自主题: Biology版 - 怎么做crosslinking?
你crosslink太久形成giant complex了吧 去google,pubmed, 或者问卖试剂的公司弄
一个protocol
W****C
发帖数: 1937
15
来自主题: Biology版 - help: crosslink co-immunoprecipitation
SCIENCE 2008 320(5882): 1496-1501
YASEMIN SANCAK et al.
The rag gtpases bind raptor and .....
浓度,时间(高了会使蛋白沉淀很多, 而且假阳性)要优化, 多抗比一抗好。
crosslinking很可能会大大降低单抗的亲和力
m****M
发帖数: 360
16
来自主题: Biology版 - DNA/oligo/primer和抗体crosslink
有哪位做过抗体和DNA conjugate的?crosslinker是什么?最多能连多长的DNA/oligo/
primer (i.e.双链,单链都可以)。连后是怎么分离没有连上的DNA,要保持抗体仍然
具有结合抗原的能力。非常感谢
s*********i
发帖数: 53
17
多谢!
我还看到有人在做reverse crosslink的那步加盐,不知道是什么作用。。
因为实验室木有人做ChIP要自己学,所以好多都不懂。。

protein
D******9
发帖数: 2665
18
我用DSG crosslink了一个蛋白的complex, 想用MASS看看有啥成分。 谢谢
A******y
发帖数: 2041
19
Invitrogen crosslinking kit i.e. the co-immunoprecipitation kit recommended
no more than 1 hour. Some abs will not work once cross-linked, the
efficiency is not a guarantee but the background is a lot less.
m******5
发帖数: 1383
20
小弟现在正在做Chip
所用抗体IP effeciency尚可
目前打算用此抗体做Chip,不过问题来了,sample crosslink, lysis, 以及dilute后
用Beads-antibody pulldown,之后用protein loading buffer煮30分钟后上SDS-page.
跑SDS-page后目的带附近什么也没有。 同一块胶的阳性对照也正常
input目的带也没有跑出来
请问有同学遇到过这种问题么?
补充说明一下,Chip所用材料是invivo knock in了一个tag的老鼠的组织。
m******5
发帖数: 1383
21
Thank you!
I did not go through the reverse crosslinking step for Chip-qPCR
What I did is I washed the dynal-beads by Ip buffer and boiled for 15 mins
in invitrogen LDS protein loading buffer.

recipe
m******5
发帖数: 1383
22
非常感谢!!!另外很不好意思这么迟才回复。
这也就是说westernblot crosslinked sample是个routine的手段来检测抗体是否work
for Chip了?
为什么常见的protocol都只要求Co-IP verify antibody呢?
l******3
发帖数: 24
23

我前几次做都没有这种问题,近几次出现这种情况。所以不知道是不是做的什么地方不
对。
而且一般protocol上说过度crosslink会导致细胞结团,影响超生shear chromatin。而
我加入formaldehyde一颠倒混匀就出现结团。
e****d
发帖数: 16
24

can we say the polymer is crosslinked if the polymer is neither soluble nor
meltable?
PS: i am working on Polymer Science. :)
y****u
发帖数: 50
25
来自主题: Macromolecules版 - 问: about polymer crosslink
what is PEI?
first you need to determine the UV crosslinkable group to use
then you need to graft the functional group to PEI.
m*******r
发帖数: 66
26
来自主题: Macromolecules版 - 问: about polymer crosslink

PEI is polyethylenimine.
It is easily soluble in water...
after crosslink, could I expect its NON-soluble in water?
t*******n
发帖数: 66
27
来自主题: Macromolecules版 - chemical or physical crosslinking?
I think the easiest way is to do the DSC since almost all the physical
crosslinking is related with teh crystallation. You can see whether there is
on emelting peak or not for the hydrophoblic block. good luck

blah)
membrane
can
m******g
发帖数: 1
28
来自主题: Macromolecules版 - chemical or physical crosslinking?
1. I think it is physical crosslinking as well.
2. Try to alter the pH of your polymer solution, to see if it can break the
association;
3. Dilute your solution since organized structure forms only above a critical
concentration.

blah)
membrane
can
e****a
发帖数: 117
29
来自主题: Macromolecules版 - photoinduced crosslinking
photoinitiated crosslinking不知道有什么常用的方法没有?谢谢
p*****r
发帖数: 75
30
来自主题: Macromolecules版 - Job Description-Crosslink
Job Description-Crosslink
Title: Project Manager-Bioelectrochemical Polymer Systems
Department: Jordan Valley Innovation Center (JVIC)
Location: Springfield, Missouri
Reports to: VP Business Development and Chief Technology Officer
Direct Reports: Technical Leaders
Interacts with: Government agencies, universities, customers and
subcontractors
Summary of Position:
Manages two teams of senior scientists in bioelectrochemical applications
research centered on (1) controlled
w********h
发帖数: 12367
31
crosslinked的最简单,只溶胀不溶解。
linear或者hyperbranched的,要看合成机理,
然后根据GPC里面对chain dimension的分析和NMR。
h******t
发帖数: 3858
32
但是crosslinked如果不溶解,怎么做GPC呢?
w********h
发帖数: 12367
33
crosslinked的最简单,只溶胀不溶解。
linear或者hyperbranched的,要看合成机理,
然后根据GPC里面对chain dimension的分析和NMR。
h******t
发帖数: 3858
34
但是crosslinked如果不溶解,怎么做GPC呢?
C*******e
发帖数: 4348
35
来自主题: Fashion版 - 推荐个缩小毛孔的护肤品。
crosslink?
什么跟什么crosslink?
难道是涂上去以后自己crosslink把毛孔堵住
还是跟皮肤crosslink?
不管哪种都听着很恐怖
f*********p
发帖数: 13
36
来自主题: Biology版 - Re: cross-link
I think Pierce has a very comprehensive guide for protein crosslinking
reagents. did you find anything on their website? I found almost every detail
that I needed for my exp.
however, crosslinking is a tricky thing in that it varies from protein to
protein, and the condition for crosslinking matters a lot too. if you are
doing it in the presence of crude cell lysate, i doubt if there is established
protocols to follow. you probably need to try different crosslinking reagent,
different concentrat
C*******e
发帖数: 4348
37
来自主题: Fashion版 - 推荐个缩小毛孔的护肤品。
没在官网上找到相关信息
不过如果真的会跟皮肤crosslink
我是绝对不会用的
个人观点
当然我很怀疑crosslink的效率
取决于用什么基团crosslink
很多交联剂都怕水
从网页上读到的信息是
这个东西治标不治本
就是让你的毛孔“立马”“看上去小了”
r****o
发帖数: 105
38
来自主题: Biology版 - Re: 请问Co-IP中的几个问题
1. 一般LYSIS BUFFER里头SDS的浓度比较低,最多只有1%,所以
不至於让蛋白质变性,破坏正常的结合能力。不过很难讲,你要
试试。如果你要用lysate做enzyme assay,那就最好不要用SDS,因为
会降低酶活性。
2. 不太清楚,常规的LYSIS BUFFER并不一定需要Sodium fluoride.
查了一下,NaF好像是一种phosphatase的抑制剂。
3. 选择HEPES还是TRIS主要是看在特定pH 附近的缓冲能力。HEPES
是一种酸,用它配pH从5到7.5都还可以,再偏硷性,比如到8,就不是
很好了。TRIS则是一种硷,Pka比HEPES大,配pH8到7之间的缓冲液,
还可以,再偏酸性就不合适了。
如果你要做crosslinking,并且用的是amine coupling的crosslinker,
就不能用TRIS,因为TRIS分子有四个氨基,很容易与crosslinker发生
耦联。
4. 非离子型去垢剂,Nonidet P-40 是最温和的一种。 Triton X-100
也可以。
5. 不太清楚,但是我觉得问题不大。只要你保
c*********r
发帖数: 1312
39
我想我明白了你说的crosslink和我说的crosslink不是一个东东。。。
我说的是把抗体和beads上的protein A/G交联起来,你可能是指把IP complex里的所有
protein crosslink吧。
不知道我说的对不对。^_^
C*******e
发帖数: 4348
40
来自主题: Biology版 - Glutathione Agarose求教
呃。。。。怎么说呢
我已经说了以前一直用Tris Buffer
这次不能用
因为要用阳离子交换柱,Tris有干扰
跟你说的crosslink有干扰相似又不尽然
(只有做primary amine基团的crosslinking才不能用Tris,crosslink不限于光做
primary
amine基团)
另外Glutathione Agarose不止sigma和GE两家
就像我原帖说的
我用的Thermo的(原Pierce)

Tris
buffer 有干扰
C*******e
发帖数: 4348
41
刺激前后的细胞抽提膜蛋白,然后IP,跑电泳,银染色。可惜没有特异性东西出来
——这个不奇怪吧,如果蛋白表达什么没有变化只是位置变化了的话
我觉得可以试一下crosslinking
好好选择spacer distance
平时蛋白均匀分布,大概距离多远
选个比那个距离小的spacer
富集的时候就应该能crosslink上了
不过如果蛋白富集区有很多种不同的蛋白的话
分析起来会比较痛苦
也可以选择带s--s的可以reverible的crosslinker
这样跑变性胶就能看
不要做MS

能有多大改
善。
g*********3
发帖数: 177
42
来自主题: Biology版 - 求助---关于nuclear protein
关于DNA-protein complex的
一般的策略是crosslink---nuclei isolation---Sonication---IP
实验室的师兄说crosslink后细胞比较像jelly,isolation的效率可能不好。建议可以
先cell lysis/nuclei isolation---crosslink,这样可能会损失一些DNA-Protein
complex,大家有见过第二种方法吗?可否给个链接,谢谢。另外,第一种isolation的
效率怎样。
谢谢各位。
s*****a
发帖数: 59
43
来自主题: Biology版 - Co-IP 如何cross-link?
The cheapest method to crosslink interacting proteins for Co-IP is to use
Formaldehyde as crosslinker. You need to find the lowest concentration of
formaldehyde in an initial test. I have been using 0.1% formaldehyde in PBS
to crosslink proteins on freshly harbested cells, room temperature for a few
minutes, and quench with 1.25M Glycine. Then lyse cells, sonicate and IP as
usual. I found this paper very useful: Optimization of Formaldehyde Cross-
Linking for Protein
Interaction Analysis of Non-... 阅读全帖
b******y
发帖数: 627
44
About UV crosslinking, many nucleic acid can be radicalized upon UV
treatment. Such radicals is highly reactive to active residuals in the
vicinity. Afterwards, digestion for MS. Abnormal peptides upon crosslinking
in comparison with those without crosslinking can tell you which peptide(s)
are close to the ligand.
g*********3
发帖数: 177
45
有点不同意见,说出来大家讨论下。
1. 如果是error bar太大了,是不是要考虑实验重做。我个人觉得replicates内部Ct在
0.3左右都可以接受,有些人要求严格的在0.1左右,否则就重做了。
2. 现在大家paper里好像也不强调biological replicate了。至少我觉得实验重复几次
才算是可靠的。
3. 如果不是实验系统本身的复杂性,控制好了以下几个因素,CHIP-QPCR的结果应该是
很可靠的:
crosslinking, sonication, Ab/Beads, reverse-crosslinking.
我一般处理CHIP-SEQ/QPCR都有大概10个sample,每个sample的Input(包括Mock 和 IP
的), Mock IP.etc,这些sample的Ct应该要接近甚至一样才算可靠,所以你应该有个标
准,自己的实验是不是很可靠了(这个实验其实用Input做啥normalize的都没有必要了
)。
至于那些error bar太大了,primer是不是可靠的,sample(尤其是input)有没有做过
dilution的standard ... 阅读全帖
k****o
发帖数: 728
46
来自主题: Chemistry版 - 请教一下agarose bead
请教一下,准备买Thermo的一种agarose beads,"NHS-activated agarose is
crosslinked, 6% beaded agarose resin with a particle size of 45 - 165 um”。
这里6% beaded是什么意思?crosslinked是什么crosslinked?
多谢!
k****o
发帖数: 728
47
来自主题: Chemistry版 - 请教一种agarose resin
准备买个NHS-activated agarose beads,指标说NHS-activated agarose is
crosslinked, 6% beaded agarose resin,这里的6%是什么意思?crosslinked是说什
么和什么crosslinked?
多谢!
b**s
发帖数: 589
48
来自主题: Macromolecules版 - [转载] what kind of polymer has this properties
Faint, Why you guys all pick PDMS?
Viscosity and elasticity mostly depend on the crosslinking density of the
target polymers. IF PDMS gets crosslinked, it's a kind of rubber! If
hydrogel-like polymers are not crosslinked, they are gelatinous materials,
just image the agaragar.
b**m
发帖数: 18
49

Correct me if I am wrong. Rubber is rubber-like because of the low crosslink
density. If the crosslink density is 0, it becomes a thermoplastic, and if the
crosslink density is high, it is a thermoset. Based on this understanding, why
a thermoplastic turns to a rubber when Tg is reached?
w********h
发帖数: 12367
50
hehe, you must be in polymer engineering.
our languages are quite different.
my understanding is this:
your crosslink density is my entanglement molecular weight Me,
the lower Me, the higher crosslink density.
when Me=M0 (monomer molecular weight), it is crosslinked (also called
thermoset). is that right?
PS Tg=100C Me=18000g/mol
PBD Tg=-100C Me=1543g/mol
PS is normally glass-like (or thermoplastic) at room temperature because of
its high Tg.
When you increase your temperature to 130 to 150C,
yo
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