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Mcl-1蛋白抗體之純化及分析 Purification and Characterization of the Mouse Mcl-1 Antibody

Mcl-1蛋白抗體之純化及分析 Purification and Characterization of the Mouse Mcl-1 Antibody.

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Mcl-1蛋白抗體之純化及分析 Purification and Characterization of the Mouse Mcl-1 Antibody

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  1. Mcl-1蛋白抗體之純化及分析Purification and Characterization of the Mouse Mcl-1 Antibody • 細胞凋亡機制，對於正常生物體個體的發展與維持佔有極重要的地位，而Bcl-2家族蛋白質則在此機制中扮演重要之調控。Mcl-1蛋白質屬於Bcl-2蛋白質家族之一員，其基因會受到IL-3/GM-CSF等細胞激素刺激而大量表現，並促使細胞存活。除了在血球細胞中具有存活功能外，Mcl-1蛋白質在胚胎發育時期亦扮演重要之角色。若胚胎發育時期缺乏Mcl-1蛋白質會導致胚胎無法著床，促使胚胎死亡。我們為了更進一步了解Mcl-1蛋白質在生理上之功能，故得先製備Mcl-1抗體血清。利用GST融合蛋白質所純化之mMcl-1蛋白當作抗原，得到mMcl-1抗體血清，再利用帶有His tag之mMcl-1作Affinity 純化，得到具專一性之mMcl-1抗體。檢測此抗體之效價發現，其用在西方點墨法以及免疫沉澱法之效果較佳。在Ba/F3細胞中，此純化過之抗體血清可辨識到兩個mMcl-1的蛋白質band。利用2D-PAGE的方式分析則使這兩個band分為五點，且均受到IL-3之誘發，但是會被Wortamannin以及SB203580所抑制，未來工作則需更進一步的釐清此五個蛋白點為何。此外，也檢測此純化之抗體是否能作切片免疫染色，以期分析mMCl-1蛋白質在老鼠胚胎發育時期表現之位置及分布。

  2. Apoptosis is essential to the normal development of animals and the Bcl-2 family proteins play an important role in this process. Mcl-1, one member of the Bcl-2 family proteins, is an immediate early gene activated by IL-3/GM-CSF and is one component of the viability response of these cytokines. In addition to a survival function in the hematopoietic cells, Mcl-1 plays a role during early embryo development as Mcl-1 deficient mice die at the peri-implantation stage. To help understand the physiological function of Mcl-1, antibody recognizing mouse Mcl-1 protein (mMcl-1) has been generated by immunizing rabbits with full-length protein derived from a GST-fusion product. An E. coli expressed His-tagged mMcl-1 has been produced for the purpose of affinity-purification of the specific rabbit serum. This rabbit serum is good for immuno-precipitation as well as Western blotting analyses. Purified mMcl-1 antibody recognizes two specific protein bands in IL-3-stimulated Ba/F3 cells. Two-dimensional gel analysis further resolves these two bands into five prominent spots, all of them are significantly induced by IL-3 but blocked by pre-treatment of cells with wortmannin, a PI3-K inhibitor. Further experiments are required to reveal the identity of these protein bands. In addition, this antibody will be tested for its application in immunohistochemistry analysis of mouse tissues .

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