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Detection and quantitation of low-abundant proteins with ECL Plex fluorescent Western blotting

O. Rönn1, M. Landström2, A. Edman-Örlefors1, and S. Edlund1
1 GE Healthcare, Uppsala, Sweden
2 Ludwig Institute for Cancer Research, Uppsala, Sweden


ECL Plex™ Western Blotting Detection System can detect proteins at picogram levels, with linearity over 3.6 orders of magnitude. Two antigens can be detected simultaneously, and differential quantitation of low-abundant proteins, such as phosphoproteins in cell lysates, can be performed.


Introduction
ECL Plex Western blotting detection system is based on direct fluorescent light detection from CyDye™ labeled antibody conjugates. The use of different fluorophores, Cy3 and Cy5, enables simultaneous detection of multiple antigens on the same blotted membrane (Fig 1). This eliminates possible loss of material from a stripping procedure, providing more accurate data. Together with a high-end imaging system (e.g. Typhoon™ scanner or Ettan™ DIGE Imager), this results in high linearity and broad dynamic range for superior detection and quantitation of low-abundant proteins.


Methods
Pure proteins or cell extracts were run on 12% Trisglycine gels. After electrophoresis, gels were blotted onto a Hybond™ ECL or Hybond-LFP membrane, and then probed with primary antibodies and secondary CyDyeconjugated antibodies according to the manufacturer’s instructions. Please see the application note Multiplex protein detection using the ECL Plex fluorescent Western blotting system (28-4015-40) (1), available at www.amershambiosciences.com, for detailed methods.


Results and discussion
Sensitivity and dynamic r
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