i***0
发帖数: 160 | 1 HI,I have a question about Western Blot. We have immunized rabbit and
obtained polyclonal antibody. using the purified antibody from rabbit serum,
we could see specific band toward our antigen. However, with ECL
luminescence, I always have a dirty background, with dots and cloudy smear
that make the image so ugly and not publishable. Do you have any suggestions
? I appreciate a lot for your help! |
|
o********r
发帖数: 775 | 2 Review
Many different tumor types have polyclonal tumor origin: Evidence and
implications
Barbara L. ParsonsCorresponding Author Contact Information, a, E-mail The
Corresponding Author
aDivision of Genetic and Reproductive Toxicology, National Center for
Toxicological Research, HFT-120, 3900 NCTR Road, USFDA, Jefferson, AR 72079,
United States
Received 7 February 2008;
revised 14 May 2008;
accepted 15 May 2008.
Available online 31 May 2008.
Abstract
Few ideas have gained such strong acceptance i... 阅读全帖 |
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l**********1
发帖数: 5204 | 3 2k USD 一支
SP1003 - Affinity Purified anti-Phosphopeptide Polyclonal Antibody - Goat
full-scale purification package - Introductory offer - $1950
如没特殊要求的话
至于特殊要求, 楼主在美国的话 直接打免费电话 到加州的人家办公室 问下吧
US office
Everest Biotech c/o Abcore
405 Maple Street, Suite A106
Ramona
CA 92065
USA
Inquiries: info (at) everestbiotech.com
Sales: usasales (at) everestbiotech.com
Technical support: support (at) everestbiotech.com
Tel: 888-320-4628 (toll free)
(from 8.00 PST to 16.30 PST)
Fax: 888-841-9041
//everestbiote... 阅读全帖 |
|
l**********1
发帖数: 5204 | 4 RE: 德艺双馨的楼主
please go to
//www.creativebiomart.net/description_10243_28.htm
You Position: Home >> Antibody >> Polyclonal Antibody >> Rabbit PAb >> Anti-Cdh5 PAb
Anti-Cdh5 PAb
Cat. No.
CPB-133RM
>
Immunogen
Recombinant mouse CDH5 protein.
Antibody Type
Rabbit Polyclonal Antibody.
Ig Type
Rabbit IgG.
Formulation
0.2 μm filtered solution in PBS with 5% trehalose
Preparation
Produced in rabbits immunized with purified, human cell-derived, recombinant mouse CDH5 extracellula... 阅读全帖 |
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M********r
发帖数: 142 | 5 pls refer http://www.nature.com/ncb/journal/v15/n4/full/ncb2702.html?WT.ec_id=NCB-201304
most important is ab.
Yi Zhang used F7452 sigma. nobody won't trust yi zhang ..
ChIP-seq sample preparation.
For Kdm2b ChIP, Kdm2b Flag-tag knock-in mESCs were fixed with 2 mM
ethylene glycol bis(succinimidylsuccinate) (Thermo Scientific) for 1 h
, followed by 10 min in 1% formaldehyde and 5 min in 0.125
;M glycine to sequence the reaction. Cells were lysed in 1% SDS, 10 ... 阅读全帖 |
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z*t
发帖数: 863 | 6 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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x********s
发帖数: 448 | 7 来自主题: Immigration版 - 审稿机会 A manuscript for a journal in analytical chemistry.
Key words: Chemiluminescence immunoassay, polyclonal antibody, magnetic
particle, organophosphorus pesticides
Need someone with approporiate background. Please send me your complete
contact info inclduing job title,working address and email, as well as some
research background info with publication list. |
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T**********e
发帖数: 29576 | 8
中国基本单克隆, 欧洲polyclonal妖娥子太多. |
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J**n
发帖数: 1224 | 9 That's not surprising at all. I will be very surprised if it was the
other way around.
Monoclonal antibody is against one specific epitype of antigen. If
it works, it works. If not (for example, under immunohistochemistry
conditions, methanol, paraglutahyde...), it won't. Polyclonal antibody
is against a great variety epitype of the antigens. Some clones of
antibody might only work for western, the others might work for immunohist,
some might work for flow, eta. Very few monoclonal antibodies ca |
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c****l
发帖数: 1086 | 10 http://www.leica-microsystems.com/products/novocastra-reagents/primary-antibodies
Look up here. I cannot remember which one exactly
but we use Rabbit polyclonal anti-Ki67, I think they probably have only one
Rabbit.I can have a look tomorrow and let you know.
Regular IHC protocol,
Mouse brain, 4%PFA fixation O/N
5um paraffin section (or 12um Cryosection)
deparaffination
ph 6.0 Citric acid antigen retrieval 100c 10 min
1 ab 4c O/N
2 ab Rm 1-2hrs
any question, let me know |
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h********n
发帖数: 4079 | 11 楼主, 你没有讲清楚整个实验的目的. 你能不能说说你为啥要在这么多细胞系里挑单克
隆呢?
总体上说, monoclonal (clonal) 和 polyclonal cell line用在实验中各有优缺点,
不能一概而论. 比如我过去的实验, 一般不用clonal cell line, 直接用FACS或者筛
选过的细胞(pool)来做实验.
如何选择取决于你的实验目的. |
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a******u
发帖数: 211 | 12 1. Int Arch Allergy Appl Immunol. 1982;68(4):377-81.
Chitin: an immunological adjuvant and a polyclonal B-lymphocyte activator.
Diamantstein T, Klos M, Osawa H, Chen ZC.
2. Arch Immunol Ther Exp (Warsz). 1991;39(1-2):127-32.
Immunoadjuvant properties of chitosan.
Marcinkiewicz J, Polewska A, Knapczyk J.
Please send the papers to e*********[email protected]
Thanks so much!!!! |
|
D**A
发帖数: 311 | 13 GFP TRAP
超级高效，省时，非抗体
POLYCLONAL ANTI GFP，CLONTECH，效果比ROCHE的好很多，但是价格稍贵。 |
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J********3
发帖数: 3151 | 14 恩，俺在以前的Lab也用CLONTECH 的POLYCLONAL Anti-GFP，效果很好，价格贵不贵就
忘了，当时买了3管，有的IP非常好，有的只能做WB。 |
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N***P
发帖数: 153 | 15 In Vivo. 2008 Jan-Feb;22(1):27-35.
Characterization of a rabbit antihuman mechano growth factor (MGF)
polyclonal antibody against the last 24 amino acids of the E domain.
PMID: 18396778 [PubMed - indexed for MEDLINE]
10个包子感谢。 |
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s******y
发帖数: 28562 | 16 rabbit polyclonal antiGFP from abcam is the best one I ever used |
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s*****g
发帖数: 7857 | 17 Cell signaling :
以前用
LC3B Antibody #2775
但它是Polyclonal antibodies
现在有
LC3B (D11) XP™ Rabbit mAb #3868
LC3A (D50G8) XP™ Rabbit mAb #4599
买了其中一个,但还没有用. |
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w**********9
发帖数: 242 | 18 俺觉得这个polyclonal origin 很山寨. 难以置信. 等大牛或教科书改了后俺跟着就是
了.
As of heterogeneity, is this you made up or you got from somewhere ? Did
you ever read Volgestin's work about tumorigenesis in spontaneous colon
cancer ?
=======================================================
3. heterogeneity
As for the heterogeneity of primary tumor, my understanding is that tumor st
arted from one cell that got original mutation and started to divide. When c
ell expanded, some of the cells accumulated other mutations. Alth... 阅读全帖 |
|
s******y
发帖数: 28562 | 19 可是有时候我们的知识并不是那么完善，敲的时候根据理论以为是最重要的
exon并不一定就真的是细胞里最重要的exon, 甚至有些隐藏的exon很多人
并不一定就知道。
所以敲完之后，必须要用northern blot and polyclonal ab WB 做验证，就是这个原因 |
|
C*****h
发帖数: 926 | 20 Rabbit Polyclonal Antibody bleeds typically yield 10-15 ml serum/rabbit
on a routine bleed and 50-80 ml/rabbit upon exsanguination. |
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j*****a
发帖数: 658 | 21 I need do IP and 1st antibody is polyclonal. Can somebody recommendate a
company with good proteinA-coupled sepharose beads? I will order soon.. GE
health has protein A sepharose CL-4B seems veryyyyy expensive, they only
have 25ml size which is ~$1395. Can someone recommendate another one with
good quality and not so expensive? Thank you guys very much. |
|
w******e
发帖数: 1187 | 22 to answer this question, let's do a "simple" math 1st:
Say you generated polyclone antibody which let's say has only two Abs -- X (
the
"nonspecific" one), Y (the "specific" one). the ratio b/w X and Y is 100:1.
the affinity of both abs against the real target P is 1nM, while the
affinity
against the homolog Q is 10nM for X, but 1uM for Y (hence the specificity
difference). You want to add excessive amount of Q to soak off Ab X so that
the final ratio b/w X and Y is 1:10 instead of 100:1, and th... 阅读全帖 |
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p******i
发帖数: 1092 | 23 计划做一个protein的tetramer，然后用tetramer做binding.
思路是：
1) express protein from 293T supernatant，purify by HIS column
2）in vitro biotinylation by BirA biotin ligase (site-specific)
3) add streptavidin-PE to make TETRAMER
请问大家觉得我这个思路可以吗？
哪位做过TETRAMER的，还请不吝赐教啊～～～多谢了～～～
另外，能请各位看看下面的内容给提点意见吗？因为克隆测过序没问题，但是现在还没
看到secretion的表达 = =
在protein的N端加上：
kozak-preprotrypsin signal peptide（for secretion，貌似这样产量会高一些，而
且可能提纯好做一些）, AviTag peptide (AviTag 是BirA ligase的识别site，可以用
来做site specific的biotinylation)
preprotrypsin ... 阅读全帖 |
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s******y
发帖数: 28562 | 24 Sigma monoclonal mouse anti-b-tubulin
Cytoskeleton INC polyclonal rabbit anti-actin (beta and gamma) |
|
f******s
发帖数: 115 | 25 TLR4 (M-16) is an affinity purified goat polyclonal antibody raised against
a peptide mapping at the C-terminus of TLR4 of mouse origin.
这个就是山羊抗鼠的了吧 |
|
s****a
发帖数: 454 | 26 the antibody will be polyclonal antibody for IP.
Shall I use IgG or serum for preclear? |
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L***s
发帖数: 133 | 27 链接打不开，
是这个么 Goat polyclonal Secondary Antibody to Chicken IgY - H&L (FITC)(
ab46969)
用上面推荐的1:250？
Thanks！ |
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D******9
发帖数: 2665 | 28 depends on your antibody again. crosslinking will destroy the epitope of the
protein. If you are using polyclonal Ab, you may have more chances |
|
b**********8
发帖数: 349 | 29 如题，10cm dish MEF cells，detect a ectopic protein(tagged with FLAG)
binding with a gene's promoter, using polyclonal rabbit anti-FLAG ab(Sigma,
Catalog Number F7425), follow the upstate ChIP kit protocol. Want to konw
how much ab should be included in each reaction. Anybody gives some tips? A
published paper with detailed information in ChIP methods using this ab will
greatly help.
Thx. |
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c**n
发帖数: 73 | 30 很久之前所以既不清楚了
但肯定是转染后过了好几天
用的是polyclonal的细胞作dilution
在dilution之前并没有经过挑阳性细胞这步
我用的是corning 的single cell cloning protocol
你google就能找到
是同一批细胞.
dilution
降低一半表达量也降低一半的细胞是同一批吗？ |
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a********n
发帖数: 844 | 31 antiboresources.com给了一个巨长的单子，也不知道哪家比较靠得住。我需要的是
polyclonal antibody,自己提供蛋白作为抗原，也考虑让公司设计合成peptide当抗原
。求推荐信誉好的公司，我好去拿个quote，谢谢！ |
|
t**a
发帖数: 255 | 32 rabbit polyclonal from covance/ Babco. Cat #PRB-106C
IHC use 1:3000,
WB use 1:1000 |
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h******u
发帖数: 602 | 33 泛泛的来说，做免疫组化是单抗(monoclonal)好还是多抗(polyclonal)好？谢谢了。 |
|
w****s
发帖数: 235 | 34 I'm having a problem to yield sufficient antibody (rab polyclonal)from
the
1st serum bleed (~6ml serum).. 总共得到了100 ug.. but based on the ELISA,
the antibody
tilter is pretty high ~100k; my intuition is that I should get much
more. so I guess either I did not X- link well
the
peptide/antigen( should check with MS though) or simply low yield is
expected for 1st bleed?
有谁做过/有经验么？ 谢谢。 |
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r******y
发帖数: 21907 | 35 最好是monoclonal的 polyclonal的也可以接受了 |
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s*******e
发帖数: 12 | 36 我们用过Cayman Chemical 的 COX-2 抗体，非常好。 不过我们是用来做Western
blot 的， 不知道你们试没试过他们家的。 是Rabbit polyclonal的， 具体的catalog
number我不记得了。 如果你需要， 我可以去实验室查了告诉你。 |
|
g*********5
发帖数: 2533 | 37 I have peptide sequence and need rabbit polyclonal antibody sevice
whcih company is good for price/quality?
my tried biosyn, covance. feel expensive, and quality so so.
thanks. |
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r***n
发帖数: 52 | 38 做IP的negative control一般用isotype control,就是和IP的antibody一样的species,
right?现在我做的IP用的是cell signaling rabbit antibody (polyclonal),
negative control用的是santa cruz的 normal rabbit IgG, 这个有很多的reference
paper引用,看文章上都很干净。但是，我做的结果就是IgG control总有protein of
interest,量和IP的一样多， 这个蛋白是用mouse Ab检测的，MW是90，所以不可能是重
链。奇怪的是，当我检测IP的那个protein (cell signaling),根据说明要4度过夜，结
果发现上次检测的IgG control的目的蛋白量减少了，但不是完全没有，因为我这两个
蛋白都是用ECL检测的，就是说上次检测蛋白的信号还在。但是我又用anti-mouse的二
抗做ECL，IgG control和IP的量又是一样。
这是怎么回事？
还有，有谁知道 cell signaling的Ab用多少... 阅读全帖 |
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r***n
发帖数: 52 | 39 做IP的negative control一般用isotype control,就是和IP的antibody一样的species,
right?现在我做的IP用的是cell signaling rabbit antibody (polyclonal),
negative control用的是santa cruz的 normal rabbit IgG, 这个有很多的reference
paper引用,看文章上都很干净。但是，我做的结果就是IgG control总有protein of
interest,量和IP的一样多， 这个蛋白是用mouse Ab检测的，MW是90，所以不可能是重
链。奇怪的是，当我检测IP的那个protein (cell signaling),根据说明要4度过夜，结
果发现上次检测的IgG control的目的蛋白量减少了，但不是完全没有，因为我这两个
蛋白都是用ECL检测的，就是说上次检测蛋白的信号还在。但是我又用anti-mouse的二
抗做ECL，IgG control和IP的量又是一样。
这是怎么回事？
还有，有谁知道 cell signaling的Ab用多少... 阅读全帖 |
|
T****O
发帖数: 407 | 40 1. Polyclonal Abs would be less specific to Ac-K9. Hypothetically, an Ab
could grab AA4-AA8 regardless antigens have Ac-K9 and Ac-K12 or not.
To get Ac-site-specific Abs you'll have to make them, affinity-purify and
confirm them with those peptides. |
|
|
i***0
发帖数: 160 | 42 I have tried increase wash volume, wash time, times of wash, adjusting
antibody concentration. Occasionally, I got a relatively clean image, but
still not clean. Someone told me I could try add some salt in PBST for wash,
and I am trying it today. I will report my result when I got it. But if you
have any great tricks, please help. Thanks |
|
h*******o
发帖数: 4884 | 43 1) try other blocking solution and block longer
try BSA, non-fat milk, or both and block at least for 1 hour
2) Reduce your 2nd antibody concentration.
3) For primary antibody incubation, incubate in blocking buffer and try
either 1-2 hr at RT or 4oc overnight, whichever one you haven't tried yet |
|
i***0
发帖数: 160 | 44 Thank you very much! Currently I am using 5% milk and 10^4 times of dilution
for secondary antibody.But I will try BSA next time. I have tried primary 1
hr at RT and O/N, and it seems better with primary 1 hr. I just hate
trouble shooting. |
|
i***0
发帖数: 160 | 45 It turns out great with 100 mM NaCl PBST washing. In addition to that I
increase wash times from 3 to 5, and rinse with fresh PBST before put the
membrane in PBST wash box. |
|
O**U
发帖数: 72 | 46 从Peptide合成到制备抗体
polyclonal,monoclonal各多少钱？公司之间的价格是不是差别挺大啊？有高手指点一
下不？
虽然没多少钱，有用的信息给个包子还是可以的。谢谢了 |
|
s******y
发帖数: 28562 | 47 大部分polyclonal anti-GFP 抗体也能识别 YFP的。 |
|
|
c****l
发帖数: 1086 | 49 这不是很正常吗，你的ko可能是in frame 的exon 1 replacement，蛋白的其他domain
还在， 如果你的抗体是polyclonal或是别exon1以外的epitope，当然可以监测到了 |
|
M********r
发帖数: 142 | 50 no no no never M2 beads.
I compared and it never works.
2 reasons
1. ChIP buffer contains SDS and SDS kills M2 beads (refer to M2 beads
handbook)
2. you never know how much antibody you used. 10 ul of M2 beads perhaps
contain hundreds ug of ab. and saturate your chromatin. you need titration,
but impossible.
you may want to try M2 antibody which is a monoclonal antibody and the same
as the one conjugated with M2 beads.
You could buy M2 antibody and F ... polyclonal antibody and use both. you
c... 阅读全帖 |
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