Details
| Original language | English |
|---|---|
| Pages (from-to) | 144-151 |
| Number of pages | 8 |
| Journal | Engineering in life sciences |
| Volume | 12 |
| Issue number | 2 |
| Publication status | Published - 26 Jan 2012 |
Abstract
This study presents and discusses the application of Cy3-labeled aptamers (where Cy3 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 (Pseudomonas 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.
Keywords
- Aptamer, His-tag, Microarray, Protein
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
- Environmental Science(all)
- Environmental Engineering
- Chemical Engineering(all)
- Bioengineering
Cite this
- Standard
- Harvard
- Apa
- Vancouver
- BibTeX
- RIS
In: Engineering in life sciences, Vol. 12, No. 2, 26.01.2012, p. 144-151.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Aptamers as detection molecules on reverse phase protein microarrays for the analysis of cell lysates
AU - Lübbecke, Miriam
AU - Walter, Johanna Gabriela
AU - Stahl, Frank
AU - Scheper, Thomas
PY - 2012/1/26
Y1 - 2012/1/26
N2 - This study presents and discusses the application of Cy3-labeled aptamers (where Cy3 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 (Pseudomonas 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.
AB - This study presents and discusses the application of Cy3-labeled aptamers (where Cy3 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 (Pseudomonas 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.
KW - Aptamer
KW - His-tag
KW - Microarray
KW - Protein
UR - http://www.scopus.com/inward/record.url?scp=84860292984&partnerID=8YFLogxK
U2 - 10.1002/elsc.201100100
DO - 10.1002/elsc.201100100
M3 - Article
AN - SCOPUS:84860292984
VL - 12
SP - 144
EP - 151
JO - Engineering in life sciences
JF - Engineering in life sciences
SN - 1618-0240
IS - 2
ER -