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GE Healthcare Ettan DIGE-Fluorescent differential protein expression and analysis system
2008-9-24
功能蛋白质组学研究的有力工具:GE Healthcare Ettan DIGE荧光差异蛋白表达分析系统
原理和应用:
    Ettan DIGE荧光差异蛋白表达分析系统在传统双向电泳技术的基础上,结合了多重荧光分析的方法,在同一块胶上共同分离多个分别由不同荧光标记的样品,并第一次引入了内标的概念,极大地提高了结果的准确性,可靠性和重复性。在DIGE技术中,每个蛋白点都有它自己的内标,并且软件全自动根据每个蛋白点的内标对其表达量进行校准,保证所检测到的蛋白丰度变化是真实的。DIGE技术可检测到样品间小于10%的蛋白表达差异,统计学可信度达到95%以上。利用Ettan DIGE技术还可以对微量(少到5μg)样本进行蛋白质组学分析,例如激光捕获显微切割(LCM)得到的样品或者很难获得的珍贵样品等。

    Ettan DIGE系统包括CyDye DIGE荧光标记物,IPGphor II/Ettan DALT电泳系统,Typhoon多功能激光共聚焦扫描仪和DeCyder差异分析软件。CyDye DIGE荧光标记物专门为Ettan DIGE系统设计,这些荧光标记物是分子量和电荷匹配的,具有信号强,光谱分开,吸收和发射峰窄等特点。这些特点使不同的CyDye(Cy2, Cy3, Cy5)标记的样品可以在同一块胶上共分离,保证所有样品在完全相同的第一向和第二向电泳条件下分离,消除实验的偏差并保证精确的胶内匹配。利用IPGphor II进行第一向分离和Ettan DALT垂直电泳仪进行第二向分离,在一块2-D胶上同时分离多达3个样品。Typhoon的光学系统经过优化,在Ettan DIGE系统的CyDye DIGE荧光标记蛋白成像中能达到高灵敏度,并且其控制软件经过优化设计采集Ettan DIGE图像。全自动多色荧光扫描功能可以一次检测多个样品并做到四色荧光一次成像,既保证了分析准确性又提高了通量。另外,Typhoon除了能检测范围广泛的荧光素外,还有经过验证的磷屏同位素检测和直接化学发光成像功能。特别开发的软件DeCyder完全自动化进行DIGE结果的多重样品间的差异比较,并通过内标对每个蛋白点和每个差异进行统计学分析。该软件全自动定位和分析在一块胶中的多重样品,并进行多块胶之间的比较分析,得到精确的蛋白丰度差异变化的计算结果。利用DeCyder软件可以得到统计学可信的结果,极大降低操作者之间的偏差,并且将手工操作时间减少到几分钟。
 采用DIGE技术,通过对不同类型,不同个体的细胞、组织、或经过不同处理和不同生长条件下蛋白质表达差异分析,在研究疾病的分子机理、分子诊断、药物作用机理、毒理学等方面都有广泛的应用。尤其是通过对各种疾病组织和正常组织进行比较,可以得到针对特定疾病(例如肿瘤)的一些标记蛋白质,得到的这些蛋白质可以用来作为疾病分子诊断的标记,或为进一步的疾病治疗以及药物开发提供有价值的信息。

    Ettan DIGE是目前蛋白质组学研究中可信度和准确率最高的技术之一,是已经经过验证并且被许多著名的蛋白质组学研究部门肯定的技术。Ettan DIGE技术已经在各种样品中得到应用,包括人、大鼠、小鼠、真菌、细菌等,主要用于功能蛋白质组学,如各种肿瘤研究,寻找疾病分子标志物,揭示药物作用的分子机制或毒理学研究等方面。
相关产品信息如下:
Reagents & Consumables For Use With the EttanTM DIGE System
Below is a complete list of all parts and conusmables that are for use with the Ettan DIGE system.
CyDyeTM DIGE Fluors
CyDye DIGE Fluor minimal dyes  
25nmol/vial to label up to 5mg of protein  
CyDye DIGE Fluor Cy2 minimal dye RPK 0272
CyDye DIGE Fluor Cy3 minimal dye RPK 0273
CyDye DIGE Fluor Cy5 minimal dye RPK 0275
10nmol/vial  
CyDye DIGE Fluor Cy2 minimal dye 25-8008-60
CyDye DIGE Fluor Cy3 minimal dye 25-8008-61
CyDye DIGE Fluor Cy5 minimal dye 25-8008-62
CyDye DIGE Fluor saturation dyes  
CyDye DIGE Fluor Labelling Kit for Scarce Samples 25-8009-83
CyDye DIGE Fluor Labelling Kit for Scarce Samples plus Preparative Gel Labelling 25-8009-84
.
2-D Electrophoresis Equipment
IPGphor  
Ettan IPGphor Isoelectric Focusing Unit 80-6414-02
Cup loading strip holder complete set 80-6459-43
IPGphor Strip Holder cleaning solution 80-6452-78
Equilibration tube set 80-6467-79
PlusOne Immobiline DryStrip Cover fluid 17-1335-01
Immobiline DryStrip Reswelling tray 7-24 cm 80-6465-32
....  
Hoefer SE600  
SE 600 Vertical Gel Electrophoresis Unit, Basic 80-6171-58
SE 600 Ruby Electrophoresis Unit, Basic 80-6479-38
Ettan DIGE Gel Alignment Guide for SE600 80-6496-29
Low Fluorescence Spacers for Hoefer SE600, 1-cm wide, 1-mm thick 80-6179-94
Low fluorescence Glass Plates for Hoefer SE 600 80-6442-14
Comb 1mm thick, 15 wells 80-6161-32
....  
Ettan DALT Systems  
Ettan DALTsix, 115 VAC/60 Hz (US) 80-6485-08
Ettan DALTsix, 220 VAC/50 Hz (Europe) 80-6485-27
Ettan DALTsix, 100 VAC, 50 or 60 Hz (Japan) 80-6494-77
Ettan DALTtwelve system. 115 VAC 80-6466-46
Ettan DALTtwelve system. 230 VAC 80-6466-27
Ettan DALT separator sheets 0.5mm 80-6467-41
Ettan DALT blank cassette Insert 80-6467-03
Low fluorescence plates with integral spacers 80-6475-58
Ettan DALT cassette rack 80-6467-98
Ettan DALT gel Caster 80-6467-22
Miscellaneous  
MultiTemp III Thermostatic Circulator, 100-120 VAC 18-1102-77
MultiTemp III Thermostatic Circulator, 220-240 VAC 18-1102-78
Electrophoresis Power Supply EPS 3501XL 18-1130-05
Power Supply Adapter set (2/pk) 18-1129-59
Wonder wedge plate separation tool 80-6127-88
2–D Electrophoresis, Principles and Methods 80-6429-60
..  
Reagents  
Urea 17-1319-01
CHAPS 17-1314-01
DTT 5g 17-1318-02
PlusOne 2–D Quant kit 80-6483-56
PlusOne 2–D Clean-Up kit 80-6484-51
PlusOne Bromophenol Blue 17-1329-01
PlusOne Tris Base 17-1321-01
PlusOne SDS powder 17-1313-01
PlusOne Glycerol (87%) 17-1325-01
PlusOne ReadySol (Acrylamide/Bis liquid Mix) 17-1308-01
PlusOne TEMED 17-1312-01
PlusOne Ammonium Persulfate 17-1311-01
PlusOne Glycine 17-1323-01
Ultrapure DMF, 250 ml 25-0089-85,
US14862-250ML
..  
Non-Amersham Biosciences Products  
DMF 99.8% anhydrous 22,705-6 Aldrich
Crew™ wipes Z23681-0 Aldrich
Crew 2™ wipes Z223681-0 Aldrich
Decon 90 cln 010 010 M.J. Patterson (Scientific) Ltd
Decon 90 (US) Catalog # 1003. Decon Laboratories, Bryn Mawr, PA 01090, Tel: 610 520-0610
Decon 90 (US) Catalog # 2901-C30. Thomas Scientific, P.O. Box 99, Swedesboro, NJ 08085, Tel: 609 467-2000
L-lysine L-5626 Sigma
pH Test strips P4536 Sigma
 
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