s******s
发帖数: 795 | 1 想研究protein A与B/C/D等的相互作用。
因为没法（或者很难）把蛋白A偶联到sepharose beads上，但是有commercially
available biotinylated protein A可以直接买（sepharose conjugated with
streptavidin当然也有卖），所以计划通过streptavidin+biotin搭桥，间接把protein
A
结合到sepharose beads上（sepharose-streptavidin + biotin-protein A），然后装
填柱子，然后apply protein B/C/D。
从来没做过avidin+biotin相关的实验，请问有朋友做过类似的么？streptavidin+
biotin的结合够强壮么？
假设 protein C可以与protein A结合，最后形成sepharose-streptavidin + biotin-
protein A + protein C。在wash和elute的时候，有特定的或者通用的条件可以
1。wash掉非特异结合的其他protein
2。特... 阅读全帖 |
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q***l
发帖数: 177 | 2 National Academy of Engineering Elects 66 Members and 10 Foreign Associates
WASHINGTON — The National Academy of Engineering (NAE) has elected 66 new
members and 10 foreign associates, announced NAE President Charles M. Vest
today. This brings the total U.S. membership to 2,254 and the number of
foreign associates to 206.
Election to the National Academy of Engineering is among the highest
professional distinctions accorded to an engineer. Academy membership
honors those who have made outstand... 阅读全帖 |
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g******6
发帖数: 782 | 3 http://www.nae.edu/56154.aspx
National Academy of Engineering Elects 66 Members and 10 Foreign Associates
WASHINGTON — The National Academy of Engineering (NAE) has elected 66 new
members and 10 foreign associates, announced NAE President Charles M. Vest
today. This brings the total U.S. membership to 2,254 and the number of
foreign associates to 206.
Election to the National Academy of Engineering is among the highest
professional distinctions accorded to an engineer. Academy membership
honor... 阅读全帖 |
|
s********p
发帖数: 1319 | 4 2010 美国心脏病协会（AHA）Scientific Sections Nov. 13-17 在Chicago进行。
与会期间，因为我自己有其他任务,蜻蜓点水，这次听的是临床方面而不是基础方面的
报道,根据Daily News, 主要述评此次年会的临床的主要热点和焦点话题：一个深刻的
危机，二个Highlights和四个Hot Topics。
一个仍然非常深刻的全球范围内的危机：
开幕式上，以神经病学家身份担任AHA现任主席的Ralph L. Sacco强调：全球范围内的
心血管疾病危机正在日益威胁着我们心脏和脑的健康。AHA 2020年的冲击目标：“改善
全美20%的心血管健康和减少20%的卒中死亡病例”将是抗击这一危机的关键。AHA定义
的“理想的心血管健康”状态，既可控的因素和健康的行为，“Life’s Simple 7 （
可控的因素：理想的胆固醇、血压和血糖水平；健康的行为：不吸烟、适当的体重、规
律的运动和健康的饮食）”，是完成这一目标的现实的、可行的、有力的武器。然而，
从Sacco领导的、始于1990年的、以3,200人为观察对象的“北曼哈顿研究（Northern
M... 阅读全帖 |
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b*****r
发帖数: 203 | 5 I'm not talking about MALDI matrix. I'm just saying if there is some matrix,
during LC run, it will be eluted out first or eluted out at different
retention time, then where is the matrix effect? |
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p*******y
发帖数: 133 | 6 HI I am doing an LC method for a drug containing a tertiary amine to couple
with MS.
Amines are known to be pretty difficult with LC.
I use TFA as ion pairing agent (A: 0.1% TFA inwater, B ACN), and the
gradient is 5% to 95% organic in 2min on a small 10cm C18 colume. I got two
peaks with similar peak area after injecting a pure compound solution in
water/acn. First eluting at solvent front and the other eluting at 2 min.
When I dissolve the compound in 0.1% TFA, I got only 1 peak at 2min. I |
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s**********8
发帖数: 25265 | 7 来自主题: MedicalDevice版 - stent http://en.wikipedia.org/wiki/Stent
Stent
From Wikipedia, the free encyclopedia
For people named Stent, see Stent (surname).
Stent
Intervention
MeSH
D015607
In the technical vocabulary of medicine, a stent is an artificial 'tube'
inserted into a natural passage/conduit in the body to prevent, or
counteract, a disease-induced, localized flow constriction. The term may
also refer to a tube used to temporarily hold such a natural conduit open to
allow access for surgery.
Contents
[hide] 1 Etymology
... 阅读全帖 |
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x*****1
发帖数: 67 | 8 如果当时有为孩子采集输血科样品可做coomb's test and elution.但关键还是测出妈
妈体内有什么抗体，因为新生儿自身不会有IgG抗体，有的也是母亲穿过去的 |
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o**4
发帖数: 35028 | 9 【 以下文字转载自 Biology 讨论区 】
发信人: oct4 (hi), 信区: Biology
标 题: 紧急求助：怎么溶解蛋白质沉淀啊？
发信站: BBS 未名空间站 (Wed Jul 25 10:46:31 2012, 美东)
我做Flag pull down，之后把elution冻在-20°过夜，今天早上解冻的时候发现蛋白质
沉淀，
我加了SDS （终浓度1%），煮了5分钟，还是没见到任何溶解，
请问该怎么溶解蛋白质啊？
用loading buffer煮一下能溶解么？
谢谢 |
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k*******3
发帖数: 450 | 10 The Chou Biomaterials Lab at the University of Texas at Tyler is accepting
visiting scholars (J1 visa) at the PhD level in the fields of Materials
Science, Chemistry/Biochemistry, Biomedical Engineering, and other related
fields. The potential candidates will be working on funded projects of
either (1) "designer surfaces" for anticoagulation coatings or (2) "drug-
eluting fibers" in wound dressing, depending on the area of the expertise.
Interested candidates should send a CV with a cover letter... 阅读全帖 |
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c********e
发帖数: 496 | 11 I heard many stories like this, three stents for coronary heart disease.
by pass/CABG is indicated in the treatment of multiple lesions (including 3)
.
the most I've heard is 7, imagine 7 stents in one heart, with one year re-
stenosis rate for one stent is 10-20% (3% for rapamycin eluting stent), what
's the total.
totally a disaster. |
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c********e
发帖数: 496 | 12 爸爸有冠心病，最近觉得胸闷，很不舒服，现在有点考虑去做支架。我们家在一个地级
市，他们说有一个大夫挺好的，可又有点不放心，想去上海做。
1.冠心病如何确诊的，是运动平板吗？
2.事实上之家并不是一劳永逸的事情，支架后的再狭窄一直是一个比较严重的问题，即
使有eluting stent之后。
3.瑞金和中山相对好一点。一般来讲如果是常见的，非疑难的病例，有一年的培训就可
以做得不错了，但是如果是很特殊的病例，比如：左主干的（cabg可能更好一点，但是
stent也能做），这个就最好去北京或者上海了。
这个手术在地级市做可以吗？depends
另外今天爸爸去做了心电图，说缺氧量其实不大，他很怕做造影，总说做了造影
就一定要做支架了，是这样吗？不能先做造影再决定嘛？另外造影是不是肯定是创伤性
的?要切开装管子进去的？我不在他们身边，帮不上忙很是着急，麻烦大家给我点建议
，太感谢了！！！
1.造影后不一定要做支架，不过不做支架，做造影的意义也不是那么大，有点矛盾哈？
2. 手术本身不大， |
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l*******t
发帖数: 33 | 13 BMS可能会造成restenosis, 所以使用率较低。在美国BMS的使用率曾经少10percent。
后来有所上升。因 drug eluting stent 需要用antiplatelets 而不用DES, 除非你经
济上有困难或你对它过敏。再说，Plavix 不是马上要成generic? 总之这决定应该是医
生结合你母亲的comorbidity 来做出，经济只是一个因素。
invented |
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y***c
发帖数: 48 | 14 俺现在研究支架，但是engineering方面的人。合作的有很多大牛级的医生。也看过动
物实验。
楼上有位说的是biodegradable的，不作数，镁啊之类的。别听他的，哈哈。
我的意见仅供参考。也没看你们的仔细情况。不知道为啥你的医生会建议bare stent，
我听到的都是bare metal stent 容易restenosis, 现在都流行drug eluting stents.
医生喜欢cipher的，但Jnj撤出市场了。我自己看到的stents觉得nitinol弹性超好。不
过听医生的吧，护士不靠谱。 |
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c******e
发帖数: 56 | 15 来自主题: Immigration版 - 急求审稿 方向：
Polymer/bio-nanopatterning
Biointerfaces
Protein adsorption on surfaces
Drug-eluting stent (in vitro & in vivo evaluation)
Drug release
Optical fiber biosensors (fabrication and test)
Surface enhanced Raman scattering (SERS)
Cancer research by FTIR
Thanks for your time and consideration!
Pls send me msg if you need, thanks!
Good luck for everyone's GC application and paper submission! |
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p*****7
发帖数: 58 | 16 1st one
Inhibition of Batrachochytrium dendrobatidis。。。
2nd one
Chromatographic behavior of co-eluted plasma compounds。。。
无影响因子
相关领域有意者请站内联系 |
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a***8
发帖数: 2433 | 17 She steps on the line every time before firing the serve,true or just
an elution? |
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b****d
发帖数: 4648 | 18 马下:)
你说elution 有几种？
sds based or low ph based right? |
|
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b****d
发帖数: 4648 | 20 that is what i used and am using...
10. Elute bound proteins by adding 90 μl 0.2 M glycine pH 2.5 (
incubation time: 60 sec under constant mixing) followed by separation.
Transfer the supernatant to a fresh tube and add 10 μl 1M Tris-base (pH 10.
4) for neutralization.
so .....could i use high ph pbs next time? instead of stupid tris?
tris is bad for mass spec |
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S**K
发帖数: 3648 | 21 哈哈
我还的做摸一下怎么elute。你去吧，等你回来 |
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l*****k
发帖数: 587 | 22 we had a lot of discussion about CS and Bio, I happened to experienced
both, but I think I like Bio much better, probably because I spent more
time on it.
running protein elution gel and you have to go through all the procedures to get to the step...
screening a genomic library and get the gene you want... doing a western...
sent out purified protein and wait for antibody to come back, and found out
no antibody in it... or doing a fluorescence labeling which will take 3 days
to finish and anothe |
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c*****t
发帖数: 1084 | 23 你可以把你的elute和beads都跑western,看看你要得条带在哪里,然后
决定下一步如何.说不定是你的蛋白被降解了 |
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t********m
发帖数: 2 | 24 I had the same problem once. No good way to improve it. Two pieces of
suggestion:
First, try different amount of thrombin, different incubation time and
different temperature.
Second, try different enzyme.
My problem was solved by adding a TEV cleavage site between the GST and my
target protein. TEV is a very sequence specific protease. My protein was
purified by glutathione column and eluted with TEV enzyme. It worked. |
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c*********e
发帖数: 1 | 25 1. Never expect to isolate a large amount Plasmid DNA from yeast.
2. What I did is:
Incubate lytic enzyme with your yeast for an hour. Then followed the
BioRad kit miniprep protocol, start from lysis solution (equal to solutionII
in classical protocol). I guess kits from other companies are also work.
At the end, elute your DNA from matrix with 20microliter of TE instead of 100
microliter. One microliter of such extract is sufficient for a bacteria
transformation, even though it's CEN plasmi |
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d*****r
发帖数: 2583 | 26 ☆─────────────────────────────────────☆
luojingjing (jingjing) 于 (Thu Jan 8 22:50:20 2009) 提到:
昨天送的，今天的数据一塌糊涂，说是测不了。
请问如何准备样品啊？
☆─────────────────────────────────────☆
ahche (啊且) 于 (Thu Jan 8 22:56:37 2009) 提到:
most of the time, the gel purified DNA is not clean enough. I always wash
twice with PE, and spin 2.5 min after PE. Then elute by H2O, and OD260
☆─────────────────────────────────────☆
luojingjing (jingjing) 于 (Thu Jan 8 23:09:03 2009) 提到:
我用QG洗了两次
PE洗了两次，每次还等了3-5分钟，
后来 |
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h*****9
发帖数: 4028 | 27 Wash one more time, eluted DNA using 1xTE |
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m****m
发帖数: 395 | 28 这次加了BME了，还是有这个现象，明明是同一个蛋白，在不同泳道上，却跑得一前一
后，虽然间隔极近，几乎就快要一直线了，但是还是有明显的前后。我用FPLC分离的，
看曲线分段取样，然后跑在不同的道上，其实肯定是同一个东西，条带很深，杂蛋白不
带HIS-TAG，之前WASH步骤应该就洗掉了，不可能结合得那么牢，最后ELUTE的时候下来
一大堆，肯定是样品，但是不知道为什么还是这样。或者跟上样量也会有关吗？比如上
得多会跑得慢点？
我是提纯了用来测结构的，所以希望尽可能纯。 |
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a***e
发帖数: 1010 | 29 how do you know your proteins are on the beads?
how about biotin competitive elution? |
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c**a
发帖数: 94 | 30 his tagged 的蛋白用nickel resin纯化出来,在elution buffer里, buffer里有
imidazole, sodium
phosphate. 一共1ml. 以后需要做activity asssy, 请问怎么保存才能不失活性?
是不是要加protease inhibitors, glycerol? 加多少glycerol? 冻-20还是-80? |
|
c**a
发帖数: 94 | 31 谢谢大家, fresh的蛋白还在elution buffer里时 就做过activity assay, imidazole不影响活性. 放4度
一个星期之后蛋白就不行了. 所以想存在低温下.
回头拜读一下从纯化到星辰. |
|
h****6
发帖数: 229 | 32 I used reversible avidin column for in vivo biotin labeled protein
purification. Elution was done with biotin. Worked pretty well. Forgot which
brand. Maybe Promega. |
|
e******x
发帖数: 9 | 33 看到这里有很多牛人都做过CHIP试验。 想请教一下大家：
（1）一般做完IP以后大家是怎么purify pulled-down chromatin DNA的？ 我看到网上
说可以
用PCR purification Kit 直接纯化，请问这里有人试过吗？
（2） 我最近才开始摸索条件，上一次实践结果，一个sonicated lysate sample 还可
以， 但另
外一个IgG control 有阳性。请问可能什么地方出现问题了呢？
（3）大家有什么好的protocol可以推荐一下啊？我只是需要做一个简单的Myc-tag的
CHIP. 抓下来
Chromatin DNA 看看几个结合位点再两个条件下有没有变化。最后的结果想用Real-
time做。上次
买了一个Active Motif 的CHIP Kit. 但发现最后的elute buffer 好像和我的SYBR Mix
不兼
容，严重影响PCR反应 （必须稀释20倍以上）。
望高人指点， 万分感谢！！！！ |
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a***e
发帖数: 1010 | 34 after elution, add >5x PB buffer from Qiagen kit, the clean up by the Qiagen
column. |
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e******x
发帖数: 9 | 35 DO I need add any carriers in the elution before I load to PCR purification
column? |
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r*********y
发帖数: 124 | 36 我做M2 FLAG IP后的蛋白在0.5mg/ml 3XFLAG 室温1小时可以很好的 洗下来（4ml 洗脱
体积)。在跑胶之前需要除去过量的3XFLAG,同时把体积浓缩到100-150ul。我先用GE
G25 gel filtration column除去3XFLAG, 然后用vivan spin 6浓缩，发现还有很多
3FLAG在样品里。
另外我的样品在浓缩之后很粘，上样的体积加大后胶就跑的很慢还smeared（我的bait
蛋白最多200ng)。所有纯化，浓缩都用同样的buffer, 20mMTris pH7.5, 150NaCL, 10%
Glycerol, 1%NP-40, 1mM DTT, 1mM EDTA.我想请教高手;
(1) 如何除去多余的3FLAG
(2)如何避免浓缩是样品过粘影响跑胶。
谢谢 |
|
a*****n
发帖数: 2835 | 37 没有必要去除FLAG吧？跑到胶里面自然就跑出去了
样品不浓缩试试？样品太粘的话说明蛋白浓度过大
bait
10% |
|
r*********y
发帖数: 124 | 38 不浓缩做WB没问题。样品里有总共2mg的3FLAG, 不除去交胶都跑不动。Bait蛋白浓度太
稀，我想 都上到一个well里，在送去做mass spec.
在 ankyrin (漂泊Boston) 的大作中提到: 】 |
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C***Y
发帖数: 61 | 39
状态，我们文章里面，
overexpression可以检测到binding，但内源的试了几次，摸索了不同buffer条件没有
成功，以前就看相关文献，一篇cell
里面可以内源检测到该蛋白和一个底物binding，但我们没有重复出来，就连
overexpression这2个蛋白，我们也没检测到
binding，哎，十分frustrated啊，coip就是很tricky。
Hopefully these tips will be helpful.
Make sue you have a very good 1st antibody. A strong antibody is capable of
pulling down weak interaction
partners.
Crosslink 1st antibody to beads and elute co-purified protein complex from beads
after washing (instead of boiling
beads) to reduce background.
Use ECL plu |
|
C***Y
发帖数: 61 | 40
from
beads
吗？
Here are the details:
Incubate 1at Ab with beads for 1 hr at RT. Wash away the unbound Ab.
Crosslink Ab to beads with Imidoester
Crosslinkers (Pierce). Incubate Nuclear extract (or whole cell lysate) with
beads-Ab.
After washes, elute the co-purified protein complex with low PH glycine
solution instead of boiling beads. |
|
v***a
发帖数: 1242 | 41 非常感谢
还有2个问题：
1）你是用什么buffer来incubate 1st Ab 和 beads的？跟wash一样的buffer吗？我
wash是用IP lysis buffer。你会preclean nuclear extract/cell lysate吗？
2）low pH glycine solution具体是多low？6.8？这个是可以直接elute然后
centrifuge了就可以上样跑胶？
with |
|
b*******H
发帖数: 830 | 42 pH2.8 PLUS 1/20 V of pH9.4, or pH3.5 elution plus 1/10 V of pH8.5 could be
better. otherwise, sample going to be diluted too much.
remember doing it at RT, and no more than 15min.
equal |
|
w******e
发帖数: 1187 | 43 I never tried myself either hehe. theoretically, there is a kd involved
(though it's very very low) so you can compete the DNA off. or some
harsh condition (heat, NaOH, etc) may elute the DNA off. don't know
whether you can still use the beads after that thou hehe |
|
w******e
发帖数: 1187 | 44 I agree:) the half-life should be ~10days, so if you add 100X excessive
biotin you might be able to recover your DNA after a month? hehe
but I thought PCR should be good? remember reading somewhere
ppl use heating for elution.
anyway, probably not worth it (though I'm indeed curious:) |
|
m**i
发帖数: 47 | 45 我看了NEB家的elution的条件是75C的Tris-HCl （pH 7.5)，也许streptavidin
denature了等凉了之后还能在refold? pierce家的让用Guanidine-HCl,肯定就没法
recycle了。 |
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T**********t
发帖数: 1604 | 46 用heating来elute是要加变性剂比如SDS的，光是加热掉不下来。正常PCR cycling上百
个循环都没事。 |
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g*****y
发帖数: 6325 | 47 买个除酶的dna kit. elute with low volume of water. |
|
a***a
发帖数: 40617 | 48 native gel摸條件摸到死
而且band太fat
用ion exchange是有好處，知道條件以後可以在很小體積elute出來，等於是
分離+濃縮
但是問題是也要先摸條件。而且你還不知道哪個是哪個 |
|
g*********r
发帖数: 9366 | 49 monomer and aggragate might elute out together
they might have very similiar pi |
|
C*******e
发帖数: 4348 | 50 谢谢
我现在的问题不是elute以后怎么分开两个蛋白
因为我们用PreScission Protease
我想的是最好能提高resin的利用率
现在连在resin上的大多是GST tag alone
严重影响了纯化蛋白的产率
而后续研究又要用大量的蛋白
而且最好是同一batch提的
现在想到也许可行的是ammonium sulfate ppt
the
seperate |
|
