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J Biomol Tech. 2010 September; 21(3 Suppl): S47–S48.
PMCID: PMC2918190

Specific Detection of Proteins by Immunoprecipitation Combined with Highly Sensitive Protein Sizing on Microchips

C. Wenz
Agilent Technologies R&D and Marketing GmbH & Co. KG, Waldbronn, Germany

Abstract

RP-85

Today, immunoaffinity is a crucial tool for the targeted analysis of proteins in complex samples. Techniques like Enzyme Linked Immunosorbent Assay (ELISA) and Western Blotting are widely used for a wide range of applications such as biomarker candidate verification in body fluids or clone selection for recombinant protein expression. Here we present a new method that combines the specificity of an immunoprecipitation approach with the high sensitivity of protein detection on microchips using the High Sensitivity Protein 250 assay for the Agilent 2100 Bioanalyzer. Initially, sample proteins are derivatized with a fluorescent dye. After incubation with the specific target antibody, the immunocomplexes are captured with Protein A/G coated magnetic beads, washed and eluted by heat denaturation in the presence of SDS. Samples are then directly loaded on microchips and analyzed automatically with the Bioanalyzer for protein size and quantity. The final on-chip analysis takes about 30 min for 10 samples and yields digital data. Together with the sample preparation steps, the total assay time is about 3 hours. Results are directly compared to standard Western blotting revealing similar sensitivity and specificities achieved with both techniques. However, the new approach compares favorably to Western blotting in terms of time-to-result, usability and labor intensity, antibody consumption and access to quantitative data.


Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities