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summer2771

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[求助] 做信號通路的，是不是要做定位才好��？

請問大家，大家一般做通路蛋白，都做了定位嘛？比如免疫熒光��？謝謝！我看很多文章也都只做了蛋白表達(dá)，沒有做定位的。但是熒光圖確實漂亮，感覺能出彩些。不知道通路蛋白是不是這樣的？

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1樓 2012-11-30 17:03:39

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laoboshihou

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感謝參與，應(yīng)助指數(shù) +1
summer2771: 金幣+8, 謝謝您的回帖！ 2012-12-01 01:05:17
summer2771: 金幣+8, ★★★★★最佳答案 2012-12-23 02:50:53

通路蛋白通常只做定量，但也有少數(shù)做定位的文章，樓主不妨一試。順便提供一篇范文供樓主參考。

FASEB J. 2009 Aug;23(8):2576-86. Epub 2009 Mar 19.
Cell-type specific activation of p38 and ERK mediates calcitonin gene-related peptide involvement in tolerance to morphine-induced analgesia.
Wang Z, Ma W, Chabot JG, Quirion R.
SourceDouglas Mental Health University Institute, 6875 Blvd. LaSalle, McGill University, Montreal, Quebec, Canada H4H 1R3.

Abstract
Tolerance to morphine-induced analgesia is a well-established phenomenon, often limiting its usefulness in the long-term treatment of pain. The mechanisms underlying tolerance are not well understood. We previously suggested a possible role for spinal calcitonin gene-related peptide (CGRP) in the development of tolerance to morphine-induced analgesia. In the present study, we demonstrate that CGRP is involved in morphine tolerance by differentially regulating the ERK-dependent up-regulation of IL-1beta, TNF-alpha, and microsomal prostaglandin E synthase-1 (mPGES-1) in astrocytes and p38-dependent up-regulation of IL-6 in microglia in the rat spinal cord. A 7-d treatment with morphine induced tolerance to the antinociceptive effect and increased phosphorylated ERK localized in astrocytes and phosphorylated p38 enriched in microglia, both effects being inhibited by blocking CGRP receptors. Interestingly, the inhibition of the ERK pathway suppressed the development of tolerance and morphine-induced up-regulation of IL-1beta, TNF-alpha, and mPGES-1. Blockade of p38 activity also inhibited the development of tolerance and morphine-induced IL-6 up-regulation. Taken together, these data suggest that chronic morphine induces the synthesis of CGRP, which in turn acts on CGRP receptors located on astrocytes and microglia to stimulate ERK and p38, respectively, leading to increased synthesis and release of proinflammatory mediators resulting in tolerance to morphine-induced analgesia.

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2樓2012-11-30 17:12:26

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henryxue

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summer2771: 金幣+4, ★有幫助, 謝謝您的回帖！說的也很詳細(xì)！ 2012-12-01 01:05:33

免疫熒光并不是不可以作為信號通路說明的方法，但是需要看合不合適。比如你的蛋白會在特定刺激下在胞內(nèi)聚集、定位到特殊的細(xì)胞器、有入核出核的變化，免疫熒光是非常直觀的說明手段，但是對于胞漿均勻分布的蛋白、對于銜接蛋白等等，這些用免疫熒光來說明就并不合適。另外，免疫熒光的抗體質(zhì)量也是決定最后圖片漂亮與否的關(guān)鍵因素之一。最后廢話一句，實驗室里有什么樣的試劑、設(shè)備以及可行的方法和技術(shù)，才是決定你實驗設(shè)計的關(guān)鍵，如果沒有好用的抗體，熒光顯微鏡也不給力，做免疫熒光反而會畫蛇添足。

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