Quantitative protein microarrays for time-resolved measurements of protein phosphorylation.
The quantitative analysis of signaling networks requires highly sensitive methods for the time-resolved determination of protein phosphorylation. For this reason, we developed a quantitative protein microarray that monitors the activation of multiple signaling pathways in parallel, and at high temporal resolution. A label-free sandwich approach was combined with near infrared detection, thus permitting the accurate quantification of low-level phosphoproteins in limited biological samples corresponding to less than 50,000 cells, and with a very low standard deviation of approximately 5%. The identification of suitable antibody pairs was facilitated by determining their accuracy and dynamic range using our customized software package Quantpro. Thus, we are providing an important tool to generate quantitative data for systems biology approaches, and to drive innovative diagnostic applications.
Korf
Ulrike
U
Division Molecular Genome Analysis, German Cancer Research Center, Heidelberg, Germany. u.korf@dkfz.de
Derdak
Sophia
S
Tresch
Achim
A
Henjes
Frauke
F
Schumacher
Sabrina
S
Schmidt
Christian
C
Hahn
Bettina
B
Lehmann
Wolf D
WD
Poustka
Annemarie
A
Beissbarth
Tim
T
Klingmüller
Ursula
U
D000818:Animals; D000918:Antibody Specificity; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D003429:Cross Reactions; D050822:Cytokine Receptor gp130 / Q000235:genetics / Q000378:metabolism; D007118:Immunoassay; D013058:Mass Spectrometry; D051379:Mice; D008807:Mice, Inbred BALB C; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000379:methods*; D011506:Proteins / Q000378:metabolism*; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D012984:Software; D013055:Spectrophotometry, Infrared; D049490:Systems Biology
D011506:Proteins; D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins; D050796:STAT3 Transcription Factor; D050822:Cytokine Receptor gp130; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3