Protein Microarrays to study Post Translational Modification Changes as a Function of Disease (Tasneem et.al.)
Complementary to the popular antibody arrays, this application uses pre-separated proteins from cellular lysates or other bio-fluids. The resulting arrays are probed with agents that can specifically detect certain post translational modifications. Differences in modifications between different disease states can therefore be highlighted.
Glycoproteins from serum samples can be enriched and separated by nonporous reversed-phase HPLC. Separated proteins are then arrayed on nitrocellulose slides and probed with multiple types of lectins using a biotin-streptavidin platform to detect glycan structures present in the glycoprotein.
Dilution series of standard glycoproteins to study lectin specificity. Printed glycoproteins were probed with biotinylated lectin followed by streptavidin conjugated to green florescent Alexafluor.
|Comparison of different glycan structures in sera from healthy patients and those diagnosed with pancreatic cancer using multi-lectin detection. Each vertical panel of spots represents a unique peak from the reversed-phase HPLC separation. Each separated glycoprotein was printed in 9 replicates to monitor variations due to printing. Within 10% variation was found for most fractions studied.|
Equipment: Nano-Plotter NP2.0/E
University of Michigan Medical Center
Department of Surgery
MSRB I, A510B
1150 W. Medical Center Drive
Ann Arbor, MI 48109-0650
Patwa TH, Zhao J, Anderson MA, Simeone DM, Lubman DM.: Screening of glycosylation patterns in serum using natural glycoprotein microarrays and multi-lectin fluorescence detection. Anal Chem. 2006 Sep 15;78(18):6411-21.
Zhao J, Patwa TH, Qiu W, Shedden K, Hinderer R. Misek DE, Anderson MA, Simeone DM, Lubman DM.: Glycoprotein microarrays with multi-lectin detection: unique lectin binding patterns as a tool for classifying normal, chronic pancreatitis and pancreatic cancer sera. J. Proteome Res. In Press