CAJ | 학술논문

高通量双向电泳是蛋白质组学的核心,双向电泳凝胶上蛋白质点的检测方法应具有灵敏度高、线性范围宽和兼容质谱鉴定等优点.采用差异凝胶电泳(difference gel electrophoresis,DIGE)技术以Cy3(1-(5-carboxypentyl)-1′-propylindocarbocyanine halide N-hydroxysuccinimidyl ester)和Cy5(1-(5-carboxypentyl)-1′-methylindodicarbocyanine halide N-hydroxysuccinimidyl ester)荧光分别标记正常和TNF-α处理细胞的蛋白质,用Cy2(3-(4-carboxymethyl)phenylmethyl)-3′-ethyloxacarbocyanine halide N-hydroxysuccinimidyl ester)荧光标记正常和TNF-α处理细胞蛋白质的等量混合样品作为内标,混合3种荧光标记的蛋白质后,在同一等电聚焦胶条进行聚焦,然后在聚丙烯酰胺凝胶上进行第二向电泳,用3种波长的激光激发扫描得到凝胶图象,DIGE中多个样品在同一条件下电泳,因而匹配率高,且引入内标使蛋白质点的检测与定量更为准确.DIGE技术与质谱相结合,实现了高通量和相对准确定量.与硝酸银和考马斯亮蓝染色结果相比较,DIGE技术具有灵敏度高、线性范围宽和不影响后续质谱鉴定等优点.
고통량쌍향전영시단백질조학적핵심,쌍향전영응효상단백질점적검측방법응구유령민도고、선성범위관화겸용질보감정등우점.채용차이응효전영(difference gel electrophoresis,DIGE)기술이Cy3(1-(5-carboxypentyl)-1′-propylindocarbocyanine halide N-hydroxysuccinimidyl ester)화Cy5(1-(5-carboxypentyl)-1′-methylindodicarbocyanine halide N-hydroxysuccinimidyl ester)형광분별표기정상화TNF-α처리세포적단백질,용Cy2(3-(4-carboxymethyl)phenylmethyl)-3′-ethyloxacarbocyanine halide N-hydroxysuccinimidyl ester)형광표기정상화TNF-α처리세포단백질적등량혼합양품작위내표,혼합3충형광표기적단백질후,재동일등전취초효조진행취초,연후재취병희선알응효상진행제이향전영,용3충파장적격광격발소묘득도응효도상,DIGE중다개양품재동일조건하전영,인이필배솔고,차인입내표사단백질점적검측여정량경위준학.DIGE기술여질보상결합,실현료고통량화상대준학정량.여초산은화고마사량람염색결과상비교,DIGE기술구유령민도고、선성범위관화불영향후속질보감정등우점.