Open AccessAptamers as detection molecules on reverse phase protein microarrays for the analysis of cell lysates
Author(s) -
Lübbecke Miriam,
Walter JohannaGabriela,
Stahl Frank,
Scheper Thomas
Publication year - 2012
Publication title -
engineering in life sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.547
H-Index - 57
eISSN - 1618-2863
pISSN - 1618-0240
DOI - 10.1002/elsc.201100100
Subject(s) - protein microarray , aptamer , protein array analysis , spotting , microbiology and biotechnology , fluorophore , antibody microarray , chemistry , dna microarray , nitrocellulose , amplicon , microarray , fluorescence , biology , membrane , biochemistry , antibody , gene , gene expression , polymerase chain reaction , physics , quantum mechanics , immunology , optics
This study presents and discusses the application of C y3‐labeled aptamers (where C y3 is indocarbocyanine) directed against the his‐tag (where his is histidine) for the detection of his‐tagged proteins on microarrays in a so‐called reverse phase assay. These types of assays are widely used tools in protein microarray technology. Up to now antibodies are usually applied as detection molecules. Here, two different spotting techniques, contact and noncontact spotting, as well as different types of slides, aldehyde‐modified glass slides and nitrocellulose membrane coated slides, were examined and compared. Through this study, we validated the importance of a high protein‐binding capacity of the microarray, and the labeling position of the fluorophore within the aptamer. Purified his‐tagged PFEI ( P seudomonas fluorescence esterase I) was used as a model system. Concentrations of PFEI ‐his as low as 30 nM were detectable. These results demonstrate the applicability of aptamers as stable detection molecules in protein assays. Additionally, the reverse phase assay was found to be suitable for the detection of PFEI ‐his in cell lysates. This might be of further interest in monitoring of protein production and purification processes.