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International Conference on Complex Systems (ICCS2006)

Reverse Phase Protein Arrays for protein quantification in biological samples

Holger Sültmann
German Cancer Research Center

     Full text: Not available
     Last modified: May 6, 2006

Abstract
Reverse Phase Protein Arrays for protein quantification in biological samples

Holger Sültmann, Christian Löbke, Christian Schmidt, Ulrike Korf, and Annemarie Poustka

German Cancer Research Center, Div. of Molecular Genome Analysis, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany

The measurement of protein abundance in biological samples is a standard procedure in molecular biology. However, currently available methods are not sufficient for precise measurements in high throughput.
To overcome this limitation, we have established the Reverse Phase Protein Array (RPPA) technology for relative and absolute protein quantification in a high throughput format. RPPAs carry serial dilutions of complex biological samples (e. g. cell lysates, body fluids), which are printed by piezo-spotting onto nitrocellulose-coated glass slides. Currently, approx. 600 protein spots can be delivered on each chip; the spot number can easily be extended to several thousand. Less than 10µl sample volume are required to produce a batch of 25 arrays. The detection of a protein of interest is performed by incubation with a specific antibody. After incubation with a secondary (near-infrared (NIR)-dye labeled) antibody directed against the Fc terminus of the primary antibody, the arrays are scanned with an infrared scanner, and signals are quantified.
We developed a NIR-based total protein quantification assay performed directly on the slide, detecting spotted proteins in a concentration range of 0.1 – 5 ng/µl. With specific antibodies, we established the determination of protein abundance and protein modifications on the arrays. Currently, the lowest amount of detectable protein is in the fg range, depending on the antibody used. Intra- and inter-chip signal variances are < 2% and 3.3%, respectively. In summary, the reverse phase protein array technology is a useful tool for large-scale determination of protein and protein modification abundance. The small sample sizes required are ideal for the high-throughput analyses required in systems biology approaches.




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