a****c
发帖数: 339 | 1 Was he talking about this paper?
http://www.sciencemag.org/cgi/content/full/318/5854/1296
I'm not in this field, so several naive questions.
1) He drove the donor's HSC out of their niches. Will the differentiated T
cells still attack the graft?
2) Will the grafted HSC attack the host?
3) Even if the grafted HSC gets adapted to the host antigens, will it
tolerate another grafted tissue from the same donor?
simply
in |
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w******e
发帖数: 1187 | 2 多谢。这种conjugate是不是必须在binding之后drug要跟ab脱离？不然岂不是
antigen既要是cancer specific, surface localized, 还要constantly
internalizing?
请教通常的release途径是什么？利用biodegradable bond？还是enhanced protease
activity in tumor region? |
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k******e
发帖数: 8870 | 3 Thank you very much in advance.
1. Understanding the focused CD4 T cell response to antigen and pathogenic
organisms
Immunologic Research
Volume 45, Numbers 2-3, 123-143, DOI: 10.1007/s12026-009-8095-8
2. T helper cytokine patterns: defined subsets, random expression, and
external modulation
Immunologic Research
Volume 45, Numbers 2-3, 173-184, DOI: 10.1007/s12026-009-8098-5
Our school only has the subscript to 1996, so I have no access to the two
papers above. thanks a lot! |
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s******e
发帖数: 370 | 4 GFP有时由于某些原因（比如antigen retrieval）需要boost一下
以前用过invitrogen的一个有Alexa488conjugate的anti－GFP，还可以。如果GFP还是
出不来就再加一个二抗上去 |
|
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c******r
发帖数: 3778 | 6 “20世纪90年代早期，根据全球疫苗研究院的报告，世界卫生组织监督了在尼加拉瓜、
墨西哥和菲律宾进行的大规模破伤风疫苗接种行动。”
这句话可不是实验，是大规模临床应用。
原文里面说：“英国医学期刊《柳叶刀》在1988年6月11日题为《世界卫生组织避孕疫
苗的临床试验》的文章中,确认了Comite Pro Vida de Mexico的发现。”
这篇文章确实存在，title是：“PHASE I CLINICAL TRIAL OF A WORLD HEALTH
ORGANISATION BIRTH CONTROL VACCINE”（Volume 331, Issue 8598, 11 June 1988,
Pages 1295-1298）
其abstract是这么写的：
“A birth control vaccine incorporating a synthetic peptide antigen
representing the aminoacid sequence 109-145 of the C-terminal region of the
β subunit of human ... 阅读全帖 |
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r***e
发帖数: 2539 | 7 你是说带有sv40 ori的质粒能被T antigen复制吧
？ |
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s******y
发帖数: 28562 | 8 Oh...I thought it was accidentally made with antigen T by virus infection or
something. Guess I was wrong.
So it was intentionally made by stable transfection? |
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h********0
发帖数: 944 | 9 查到的资料有的说是一回事，有的文献却分开列这两个抗体，高人指点一下 |
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b******s
发帖数: 1089 | 10 Sorry for English. Can't input chinese in the lab.
Nowadays, people choose to fix samples by high pressure freezing and freeze
substitution more and more. There is no question that this method is elegant
to keep the cellular structure and keeping the antigen active for immuno-EM
.
I read many discussions in EM mail group talking about their papers got
rejected because they used the conventional chemical fixations. It seems
people in EM field have different opinions about requirement of using hi... 阅读全帖 |
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s****9
发帖数: 932 | 12 I do not know. I assume that DC is very good at internalizing antigens and
bounded Ig can be easily internalized before successful ADCC occurs.
Similarly, I never see any reference depleting macrophages in vivo using mAb.
In addition, if CD11c-DTR cannot be used and macrophages play little role in
your system, you might consider clodronate liposome. High dose of CLL
depletes macrophages and most dendritic cells. |
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S*****s
发帖数: 287 | 13 我还以为作者里面有 Harry Potter，一高兴就重复了一下 lz 的 data，发现结果完全
是 Pseudo-positive。Pubmed 现在能搜到的含有 Harry Potter 的文章一共三十二篇
，全部贴在这里了，基本上都是在摘要或者标题里提到 Harry Potter 的。这也是
fans 多的表现吧。
1: Bellomo R, Morimatsu H, Presneill J, French C, Cole L, Story D, Uchino S,
Naka
T, Finfer S, Cooper DJ, Myburgh J; SAFE Study Investigators and the
Australian
and New Zealand Intensive Care Society Clinical Trials Group. Effects of
saline
or albumin resuscitation on standard coagulation tests. Crit Care Resusc.
2009
Dec;11(4):250-6. ... 阅读全帖 |
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o****e
发帖数: 1011 | 14 用比如NaHCO3/Na2CO3 buffer coating（antigen or antibody）完，甩掉后，
一定要洗，还是可洗可不洗？
加完blocking solution，block完，甩掉后，
一定要洗，还是可洗可不洗？
感觉block完后，似乎可以不洗，反正残余的可以继续block些空位？ |
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j*****a
发帖数: 658 | 15 I am not sure if I understand you well...I thought the difference of non-
ionic (NP40, Triton X100) and ionic (SDS, sodium deoxycholate) is non-ionic
lysis buffer doesn't denature protein. Some of the antibody that recognizes
only non-denature antigen (original state, you might want to say) so that
you can't get good results if you use denature lysis buffer (SDS) with these
antibodies. In anothe word, non-denature lysis buffer is more safe. Am I
wrong?
So I am confused with what you said about "... 阅读全帖 |
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n***3
发帖数: 663 | 16 Immunol Rev. 1995 Jun;145:5-31.
Liposomes as carriers of peptide antigens: induction of antibodies and
cytotoxic T lymphocytes to conjugated and unconjugated peptides.
Alving CR, Koulchin V, Glenn GM, Rao M.
please send the paper to e********[email protected] |
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n***3
发帖数: 663 | 17 Immunol Rev. 1995 Jun;145:5-31.
Liposomes as carriers of peptide antigens: induction of antibodies and
cytotoxic T lymphocytes to conjugated and unconjugated peptides.
Alving CR, Koulchin V, Glenn GM, Rao M.
please send the paper to e********[email protected] |
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r***e
发帖数: 2539 | 18 是说rabbit anti ki67吗？
vector lab的rabbit monoclonal不错。
这个抗体能认识human/mouse antigen。 |
|
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C******8
发帖数: 602 | 20 听版上人说做CoImmunoprecipitaion用magnetic beads可以减少nonspecific
手里刚好有active motif ChIP kit里的magnetic protein G beads
我可不可以用这个beads conjugate antibody，然后去拽cell lysate的antigen呀。心
里想差别也就是ChIP里protein先crosslink了DNA嘛。。。
washbuffer还有elution buffer有什么要注意的么？Elution难道直接加sds sample
loading buffer煮一煮，dissociate下来protein到buffer里然后load？
如果新买其它beads还得等几天，试试可不可以呀？有没有前人经验。。。 |
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s********n
发帖数: 248 | 21 我实验需要培养B6和一种mutant老鼠的Bone marrow macrophage,然后比较细胞的对LPS
,IFN beta， gamma等stimuli的反应区别，以及antigen presentation to CD4+ T
cell，nitro oxide production还有phagocytosis的区别。
我原来的protocol就是把bone marrow放在有M-CSF（L-Cell media）的media里养6-7天
，replate以后就用。后来隔壁实验室的老教授说，你这样养7天，得到的细胞其实更接
近于monocyte,还需要把细胞在no-M-CSF media里养2-3天，才能得到real macrophage
。可是我在文献中查不到这种说法的依据。
我老板原来是做T cell的，也不太清楚这个问题。
大家谁做过Macrophage啊？是不是如这个老师说的？还是说不同实验目的，培养方法也
不一样？
多谢了先！ |
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H****s
发帖数: 301 | 22 Usually people achieve this by phage/yeast antibody display technology. You
need to have a very good antibody library and yeast/phage library panning
capability. Once you have a pool of selected binders, you can screen them by
ELISA. In your case, two ELISA can be performed in parallel, one coated
with your target protein, the other coated with negative target protein. The
ELISA signal will give you a rough idea about target specificity. If you
have access to flow cytometry, combining flow sorti... 阅读全帖 |
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w******e
发帖数: 1187 | 23 我对做抗体一窍不通，谁能给我讲讲，是不是随便找一段peptide都能当antigen用?
难道构象一定能跟protein上的structure一致吗？那段peptide是不是直接合成就完了？
我phd lab做ab都是用一个domain做的，如果15AA的peptide就行，那省事多了 |
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w******e
发帖数: 1187 | 24 请教适合用作antigen的peptide都有什么criteria？多谢！ |
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w******e
发帖数: 1187 | 25 没理解。用A有B没有的部分做antigen不行吗？ |
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m******5
发帖数: 1383 | 26 google
Large T antigen NLS
one of the most commonly used NLS sequence |
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n***3
发帖数: 663 | 27 J Neuropathol Exp Neurol. 2002 Jul;61(7):614-22.
Neutralizing antibodies to IL-18 ameliorate experimental autoimmune neuritis
by counter-regulation of autoreactive Th1 responses to peripheral myelin
antigen.
Yu S, Chen Z, Mix E, Zhu SW, Winblad B, Ljunggren HG, Zhu J.
Please send the paper to e*********[email protected]
Thanks! |
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w******e
发帖数: 1187 | 28 Methods Mol Biol. 2009;504:385-98.
Using RNA aptamers and the proximity ligation assay for the detection of
cell surface antigens.
Pai SS, Ellington AD.
email: q************[email protected]
Thank you!! |
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a****d
发帖数: 1919 | 29 CHIP assay 最早是Ron Mckay建立的吧，称作 Mckay assay.
An immunoassay for the interaction between an SV40 T antigen related protein
and DNA.
J. Mol. Biol. 1981
至于在哺乳动物细胞里面用类似方法，是否尚永丰是第一人，就不清楚了。 |
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a****d
发帖数: 1919 | 30 CHIP assay 最早是Ron Mckay建立的吧，称作 Mckay assay.
An immunoassay for the interaction between an SV40 T antigen related protein
and DNA.
J. Mol. Biol. 1981
至于在哺乳动物细胞里面用类似方法，是否尚永丰是第一人，就不清楚了。 |
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n*********m
发帖数: 38 | 31 Look his publication
Conversion of D-ribulose 5-phosphate to D-xylulose 5-phosphate: new insights
from structural and biochemical studies on human RPE.
Liang W, Ouyang S, Shaw N, Joachimiak A, Zhang R, Liu ZJ.
FASEB J. 2011 Feb;25(2):497-504. Epub 2010 Oct 5.
PMID:
20923965
[PubMed - indexed for MEDLINE]
Related citations
2.
Structural basis for the inhibition of human 5,10-methenyltetrahydrofolate
synthetase by N10-substituted folate analogues.
Wu D, Li Y, Song G, Cheng C, Zhang R, Joac... 阅读全帖 |
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h*******o
发帖数: 4884 | 32 Are you sure it is the fixation problem?
Have you tried with antigen retrieval. |
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g****0
发帖数: 425 | 33 an antibody good for western-blotting is not necessary a good one for IHC or
IF. You can try it on cell culture first. How about IP with KO as control?
It is definitely a way to go try different antigen retrieval methods.
It is also useful to try different blocking reagents. |
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h********n
发帖数: 4079 | 34 各位回帖说得真好啊. 我也乱说一些我的做法.
1. what is the promoter? if you know the promoter, you can guess whether it
is a tissue specific gene.
2. what cell type in pancreas express this gene, epithelial or other?
immuno staining should answer this question.
3. select cell lines based on answer to question 1 and 2
4. take highly malignant cancer cell lines, overexpress this gene by
transient transfection, cell viability assay, tumorigenecity (in vitro and
in vivo) assay and invasion assay should be used. Growth... 阅读全帖 |
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e**s
发帖数: 513 | 35 Ann Bot (2011) 107 (7): 1127-1140. doi: 10.1093/aob/mcq243
Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication
and cell cycle regulation
Thanks！ |
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s******y
发帖数: 28562 | 36 有这个可能。他们在文章里对那些细胞进行了expression profiling.
你要是有这个经验的话能不能去瞅瞅哪个基因比较可能是antigenic的？
specific |
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j*b
发帖数: 341 | 37 This argument is not a big deal. even certain type of cancer can trigger the
immunoreaction by expressing some testis-specific antigens.iPS is not
equivalent to ES cells, and different lines of iPS have their unique
differentiation potentials.
just don't understand why nature published so many so-so paper on ips cells.
|
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j*b
发帖数: 341 | 38 The terotoma formed by ES and iPS may have a lot of differences,but the in
vitro differentiated cells from iPS may not express those "antigen" any more.
Overall, I think the discovery is interesting, but the interpretation is not
that convincing, even a little bit misleading (for publication purpose?)
Self |
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D*a
发帖数: 6830 | 39 Zhou H, Morotti RA, Profitt SA, Langston C, Wert SE, Whitsett JA, Greco MA
Expression of thyroid transcription factor-1, surfactant proteins
, type I cell-associated antigen, and Clara cell secretory
protein in pulmonary hypoplasia.
Pediatr Dev Pathol 2001, 4: 364-371.
多谢！ |
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d*p
发帖数: 534 | 40 T 细胞是有抗原特异性的，这个方法虽然能够增加T细胞和抗原的接触，但是不能特异
地把肿瘤抗原特异性的T细胞吸引过来，而是所有的T细胞，因此虽然能够增加一些效果
，但是增加的幅度有限。另外需要提到的一点是肿瘤诱导的整个免疫抑制环境，目前大
部分的治疗仍然是在inflammatory这样一个抑制大大环境下治疗，效果自然会打很大折
扣。肿瘤和病毒长期感染导致的发炎其实很相似，病毒特异性的免疫细胞在这个
inflammatory环境下大部分都存在，但是都不工作。
T cell adoptively transfer应该是特异性最强的，在临床也有不错的效果，目前需要
解决的是提高这些移植T cell的存活时间和功能，目前体外处理的方法还存在很大的空
间可以改进。移植的T cell通常是病人外周血分离出来的或者肿瘤侵润的T cell这两种
，通过体外用肿瘤抗原扩增，或者通过病毒载体转入特异行的chimeric antigen
receptor， 再移植回病人。病毒载体有lymphoma的风险，对于晚期病人来说，这个风
险可以忽略的，另外现在suicide gene也已经做出来了，能在24-48小时... 阅读全帖 |
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f******s
发帖数: 288 | 41 我觉得只要有一点免疫源就会有连锁反应，应该和量的多少没有关系。
但是好像又没有这方面的paper ？ 。。。 |
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c*****i
发帖数: 1392 | 42 还用什么paper，随便找本免疫教科书基本都有。immunogen的量太低是不能激起免疫反
应的。一般情
况是随着immunogen量的增加，免疫反应增强，到一定量后不再增强，然后再增加，免
疫反应减弱。 |
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f******s
发帖数: 288 | 43 说的是，光查paper了，没想起书来，确实是这么说的。谢啦
但是我想知道的其实就是那个 “到一定量后不再增强” ，那个量的范围有多大。不过
这应该是根据不同immunogen和不同个体决定的。 |
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f******s
发帖数: 288 | 44 我对免疫的知识非常贫乏哈。
我觉得有意思的是，实际上如果免疫源给的太多，也不会有反应，而是当量降低的时候
，反而会出现response。
到底这个给多给少，怎么能确定？比如在一种动物身上试出来的大体倍数（比如加
1000没反应，给了10就有反应了），不说绝对量，而是说这个比例倍数，能大体预计出
其他物种类似的 response 么？ |
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w******e
发帖数: 1187 | 45 http://www.nature.com/nbt/journal/v29/n6/full/nbt.1856.html
phage display是好东西。。。and又见next-gen seq in directed
evolution。。。well只做一个round的话不知还该不该叫evolution lol
不过这篇paper的pos control够单薄的。
Immune responses targeting self-proteins (autoantigens) can lead to a
variety
of autoimmune diseases. Identification of these antigens is important for
both diagnostic and therapeutic reasons. However, current approaches to
characterize autoantigens have, in most cases, met only with limited success
. Here we pres... 阅读全帖 |
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i*****g
发帖数: 11893 | 46 这个东西就是engineering思路，不幸的是，碰上了生物这个系统，死
T B DC细胞，不就是这么出来的么，antigen-receptor---effector cascade--clonal
growth--detection under microscope
体外能完成这个么？显然不能，
体内的有一些，如Y2H，FRET，
但技术都不好用，所有生物技术，大概除了分子克隆，都是通用性很差的 & low
efficiency |
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F**********6
发帖数: 90 | 47 I usually use 6XHIS tagged fusion protein (200-300aa) as an antigen to
immunize rabbit and at least 2 fragments are used for each gene.
So far I've got immunostaining-grade antiserum for several genes. |
|
j****x
发帖数: 1704 | 48 一般公司都可以提供rational antigen design的服务，从序列中特征性选取多肽片段
然后交联到BSA等载体蛋白上再制备单抗或多抗。如果目标蛋白是有三级结构信息的，
这种表位预测+多肽免疫的成功率很高，一般在70%-80%以上。即便没有高级结构信息，
有经验的设计分析人员也可以将成功率控制在50%左右，一般而言选取2-3条多肽序列就
能至少有一个能制备出不错的抗体。我们这里有core facility专门提供这种服务，这
个技术本身应该说很成熟了。
自己原核表达纯化蛋白的部分片段自然也是一个选择，以前大部分人都不纯化而只是切
胶直接免疫，取决于你要做什么样的staining了，要求抗体特异性好的，就不建议这么
做了，光做western的话还凑合。 |
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c**********5
发帖数: 653 | 49 How about expression SV40 Large T-antigen (proto-oncogene) after Pcmv |
|
v*******3
发帖数: 119 | 50 CHIP已经作了块3个月了,
全程基本自己摸索
用的是cell signal chip kit及抗体,新鲜
问题一直在:
预实验作了很多,IgG (negative control) and H3(positive control) 有显著差异,
但是其它磷酸化组蛋白与转录因子均与IgG类似或者持平或更低
2. 实验用的是 1% formadehyde固定10分钟,全程按照kit说明书来,而且适当改进重要
步骤(如 increase cross link reverse time, 减少洗得次数)
3. 用的都是全都是cell signal 抗体, 并在非 cross link情况下，用WB证实可用
4. sonication后电泳证实DNA片断200-1000 bp间,符合要求，无气泡
所以现在我自己的猜想:
1. cross link mask the epitope of antigen, resulting no antibody binding
2. 1% formadehyde is too low, should increase to 1.5 or 1.8 %(... 阅读全帖 |
|
