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A new phosphospecific cell-based ELISA for p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, protein kinase
H H Versteeg1, E Nijhuis, G R van den Brink
1Laboratory for Experimental Internal Medicine, G2-130, Academic Medical Centre, Meibergdreef 9, NL-1105 AZ Amsterdam, The Netherlands.
Abstract:
Assaying activation of signal transduction is laborious and does not allow the study of large numbers of samples, essential for high-throughput drug screens or for large groups of patients. Using phosphospecific antibodies, we have developed ELISA techniques enabling non-radioactive semi-quantitative assessment of the activation state of p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, protein kinase B and the transcription factor cAMP-response-element-binding protein (CREB) in 96-well plates. This assay has been termed PACE (phosphospecific antibody cell-based ELISA) and was used successfully for both adherent and suspension cells. Various stimuli induced dose-dependent enzymic activity of which the kinetics closely correlated with those measured via classical methodology. Using PACE we have now characterized for the first time the concentration-dependent effects of various inflammatory prostaglandins on CREB phosphorylation in macrophages. PACE is a straightforward and novel technique enabling the large-scale analysis of signal transduction.
Insights
A new assay, PACE (phosphospecific antibody cell-based ELISA), allows for the large-scale analysis of signal transduction pathways. This method enables high-throughput drug screening and patient studies by assessing protein activation in 96-well plates.
Area of Science:
- Cellular signaling and molecular biology.
- Biochemical assays and high-throughput screening.
Background:
- Assessing signal transduction activation is typically laborious, limiting large-scale studies.
- Current methods are not suitable for high-throughput drug screening or analysis of large patient cohorts.
Purpose of the Study:
- To develop a novel, high-throughput method for assessing signal transduction activation.
- To enable semi-quantitative, non-radioactive assessment of key signaling proteins.
Main Methods:
- Development of a phosphospecific antibody-based ELISA technique, termed PACE (phosphospecific antibody cell-based ELISA).
- Application of PACE in 96-well plates for analysis of adherent and suspension cells.
- Validation of PACE by correlating kinetics with classical methods.
Main Results:
- PACE successfully assessed activation of p42/p44 MAPK, p38 MAPK, protein kinase B, and CREB.
- The assay demonstrated dose-dependent enzyme activity in response to various stimuli.
- PACE was utilized to characterize prostaglandin effects on CREB phosphorylation in macrophages.
Conclusions:
- PACE is a straightforward and novel technique for large-scale signal transduction analysis.
- This method facilitates high-throughput drug screening and patient sample analysis.
- PACE enables efficient characterization of cellular signaling pathways.