Microchip CGE linked to immunoprecipitation as an alternative to Western blotting.
A novel approach for protein identification is presented, which combines the specificity of an immunoprecipitation approach with the sensitivity of protein detection in microchip CGE. This method involves derivatization of the sample proteins with a fluorescent dye, target protein isolation with specific antibodies and Protein A coated magnetic beads, and automated sizing and quantification of the eluted samples on microchips. The performance of the new technique was demonstrated with glutathion-S-transferase- and polyHistidine-tagged target proteins in an Escherichia coli background. A specific detection of target proteins was possible down to 0.001% or 1 ng target protein in a background of 100 microg E. coli protein. With this approach, proteins ranging from 10 to 220 kDa could be identified with a panel of different target-specific antibodies. The reproducibility of the method was very similar to standard microchip CGE methods. In a direct comparison to Western blotting, a similar sensitivity and specificity of both techniques was observed. 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.
Wenz
Christian
C
Agilent Technologies R&D and Marketing GmbH & Co. KG, Waldbronn, Germany. christian_wenz@agilent.com
Rüfer
Andreas
A
D015153:Blotting, Western / Q000379:methods*; D029968:Escherichia coli Proteins / Q000032:analysis; D005453:Fluorescence; D005982:Glutathione Transferase / Q000032:analysis; D047468:Immunoprecipitation; D056656:Lab-On-A-Chip Devices
D029968:Escherichia coli Proteins; D005982:Glutathione Transferase