( P O. Rönn 1...)Detection and quantitation of low-abundant proteins with ECL Plex fluorescent Western blotting

O. Rönn¹, M. Landström², A. Edman-Örlefors¹, and S. Edlund^1
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 range
A model system, with two-fold serial dilutions of human apotransferrin and actin (not shown), was used to optimize system performance (Fig 2). Protein could be detected down to 1.2 pg, giving linearity over 3.6 orders of magnitude.

This result was also dependent on a membrane with low background fluorescence at wavelengths suitable for the fluorophores. Both a nitrocellulose membrane (Hybond ECL) and a newly developed PVDF membrane (Hybond–LFP) were found to meet these expectations.

Detection and quantitation of a phosphoprotein
Multiplex detection of proteins often suffers from cross-reactive signals between antibodies or dyes. This is addressed with the ECL Plex system through thorough optimization of components and protocols (Fig 3).

TGF-βis a potent growth factor stimulating a number of cellular responses including growth inhibition, cell differentiation, and apoptosis. The TGFβ–mediated phosphorylation of p38, measured in human T293 epithelial kidney cells, showed proportional increase when compared and quantitatively related to actin (Fig 4).

Conclusions
Western blotting is a well-established technique. The most common application is based on chemiluminescence, for example the GE Healthcare ECL™ product line (2, 3). As a new member in that family, ECL Plex adds a broader dynamic range and the ability to detect more than one protein on the same blot with high sensitivity and specificity. These features complement the chemiluminescence-based products, offering better quantitative data and shorter processing time. CyDye technology also offers longer lasting signals for storage and rescanning. Low-abundant proteins are readily detected in cell lysates, and activation of signal transduction pathways can be measured.

References
1. Application note: Multiplex protein detection using the ECL Plex fluorescent Western blotting system, GE Healthcare, 28-4015-40, Edition AA (2005).

2. Harper, D. R. et al. Protein blotting: ten years on. J. Virol. Methods 30, 25–39 (1990).

3. Paladichuk, A. How the Western was won: a profile of tools and kits available for Western blotting. The Scientist 13, 18–21 (1999).

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