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- 实验准备阶段，求助！！！！
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各位老师，小弟硕士在读，实验准备中，欲用western－blot检测新西兰兔的骨髓间充质干细胞表面的缝隙连接成分蛋白Connexin43，不知购买何种一抗。有用过的老师请您指教。
有一公司给我发了产品资料，如下：
Qty: 50 μg/200 μl
Rabbit anti-Connexin 43
For Research Use Only
Catalog No. 71-0700
Lot No. Rabbit anti-Connexin 43
FORM
This polyclonal antibody is supplied as a 200 μl aliquot of affinity purified antibody at a concentration of 0.25 mg/ml in 10 mM
phosphate buffered saline, pH 7.4, containing 0.1% NaN3.
IMMUNOGEN
Peptide corresponding to a segment of the 3rd cytoplasmic domain (C-terminal portion) of rat connexin 43.
SPECIFICITY
This antibody reacts with connexin 43. On Western blots, this antibody detects a 43 kDa protein with mouse brain, rat brain,
and human smooth muscle cell extracts. Reactivity with other connexin types has not been observed on Western blots (note:
samples were not boiled prior to running SDS-PAGE). Positive immunohistostaining is seen with mouse heart tissue.
Reactivity of this antibody with connexin 43 is independent of phosphorylation status.(17)
USAGE
Working concentrations for specific applications should be determined by the investigator. Optimal dilutions will be affected
by several factors, including secondary antibody affinity, antigen concentration, sensitivity of detection method, temperature
and length of incubations, etc. We recommend the following ranges as starting points for this product.
ELISA: 0.1-1.0 μg/ml
IHC (frozen sections(15-17)): 1-5 μg/ml
IHC (paraffin-embedded*): 1-5 μg/ml
Western Blotting(17): ~2-4 μg/ml
* Paraffin embedded sections require a heat-induced epitope retrieval (HIER) step. Please visit our website or contact
Zymed for procedure. Tested on mouse heart tissue.
STORAGE
Store at 2-8°C for up to one month. Store at –20°C for long term storage. Avoid repeated freezing and thawing.
BACKGROUND(4,5)
Intracellular communication mediated by gap junctions plays an important role in a variety of cellular processes including
homeostasis, morphogenesis, cell differentiation, and growth control. Gap junctions are transmembrane channels that serve
to directly link neighboring cells by mediating the exchange of low-molecular weight metabolites, ions, and second
messengers. Gap junctions are formed by the interaction of connexons or hemichannels on adjacent cells. The connexon
itself is composed of a hexameric assembly of proteins referred to as connexins. Connexins are highly homologous proteins
encoded by a multigene family. The connexins exhibit similar structural features which include a cytoplasmic amino terminal
region, four transmembrane domains, two extracellular loops, and a carboxy-terminal cytoplasmic tail of varying length(2,3).
Comparison of the amino acid sequences of the various connexin family members indicate that the two areas of greatest
divergence amongst the connexin family members are the intracellular loop connecting the second and third transmembrane
segments and the carboxy-terminal tail. These domains are thought to mediate connexin-type specific properties including
phosphorylation, responses to gating stimuli, as well as assembly and membrane turnover. Modulation of gap junctional
communication can be achieved by multiple mechanisms and can occur very rapidly or over a period of several hours. These
mechanisms include alterations in transcription, translation, stability, postranslational processing (especially
phosphorylation), gating, and insertion or removal from the plasma membrane(7). Interestingly, reduction or alterations in the
levels or types of connexin expressed in a given cell type has been found to correlate with tumor progression and
metastasis(7,9).
说明中没提到可用于新西兰兔，偶一直不敢订货.
各位，拉兄弟一把吧!!!!!!
标签：
×参考诊断
兄弟你好，这个抗体，我是没有用过，不过谈下我的感觉。“western－blot检测新西兰兔的骨髓间充质干细胞表面的缝隙连接成分蛋白Connexin43”，1.首先你要知道做western要多少蛋白量才够？根据我及我的同学做western的经验，至少一个6孔板的细胞吧；2.而对于可能表达丰度并不高的表面的缝隙连接成分蛋白Connexin43（这个我确实不知道，干细胞是会表达缝隙连接成分的吗，不是我的研究方向，相信你看了很多文献的，^_^），它的含量如何，会不会像ACTIN之类的那么高呢，所以是否需要更多的细胞才能检测出差异？以上拙见，敬请指正。
此外谈一个发帖子的要求，希望大家共同爱护我们的论坛，不要重复发帖，因为网站的办理据我所知确实不容易，希望珍惜我们的空间，爱护我们共同的家园，这不是我的工作，只是道义上善意的提醒，希望谅解。
- 我虽不在江湖，但江湖中有我的传说。 向大家介绍 http://www.turningtechnologies.com/
