生物素标记钙调素用于植物钙调素结合蛋白的检测

Detection of Plant Calmodulin-Binding Proteins with Biotinylated Calmodulin Probe

用生物素标了己了花椰菜ＣａＭ。生物素标记的ＣａＭ具有与天然ＣａＭ相似的Ｃａ２＋依赖电泳特性，可激活ＣａＭ依赖性磷酸二酯酶，能够检测出５０ｎｇ的磷酸二酯酶。利用它建立了检测植物ＣａＭ结合蛋白的生物素－覆盖法（Ｂｉｏｔｉｎ－ｏｖｅｒｌａｙ）并证实酶标亲和素可与胡萝卜愈伤组织内６４ｋＤ蛋白质非特异结合，因此将此法运用于植物材料时必需设置酶标亲和素处理的对照。用生物素－覆盖法检测胡萝卜愈伤组织形成过程中的ＣａＭ结合蛋白时可检出２，４－Ｄ诱导的ＣａＭ结合蛋白。

Purified cauliflower calmodulin(CaM) was biotinylated by biotinamidocaproate N--hydroxysuccinimide ester. The biotinylated CaM retained resemble calcium--dependent electrophosphoretic charecter with nature CaM andits biological activity in activating CaMdependent phosphodiesterase (PDE ),and it was able to detect as little as 50ng of PDE. A biotin--overlay methodfor the detection of plant CSM-bindingproteins with biotinylated CaM andavidin-Peroxidase was developed. Theevidance that avidin-peroxidase couldnon--specificaly bind to a 64kD proteinin carrot callus was found, so the control treatment must be set to eliminatenon--speific binding when using thismethod to detect CaM binding proteinsin plant materials. Using biotin--overlaymethod. we detected a CaM bindingprotein induced by 2, 4--D during thecallus formation of carrot explants.