No abstract

We have developed Dissociable Antibody Microarray (DAMA) staining technology that provides a new approach to the global analysis of protein subcellular localization (SCL) in fixed cells. We have developed and optimized this technology for protein SCL profiling, generated ChipView, a program for management and analysis of molecular image database, and utilized the technique to identify proteins with unique SCL in breast cancer cell lines. We compared the SCL profiles of 325 proteins among nine different breast cell lines, and have identified one protein, Cyclin B1, with distinctively different SCLs between normal and cancer cell lines. With classic individual immunostaining, Cyclin B1 was confirmed to localize to the cytoplasm of seven breast cancer cell lines and in both cytoplasm and nuclei of two normal breast cell lines, and to have higher expression levels in the cancer cell lines tested.

Background: Dissociable Antibody MicroArray (DAMA) staining technology is a new approach for global analysis of protein expression and subcellular localization profiles in fixed cells. We have developed the technology and demonstrated its application on identifying differentially expressed proteins in breast cancer cells. We have determined the subcellular localization profiles of 360 proteins in breast cells and identified a protein with unique subcellular localization in breast cancer cells, by combining data from four 96-antibody format arrays. In this report, we have further developed the technology to determine the subcellular localization profiles of 400 proteins simultaneously from single chip, in a 400-antibody format. Method: We have determined the subcellular localization profiles of 400 arrayed antibodies in two normal prostate cell lines (PWR-1E and PZ-HPV-7), and three cancer cell lines (Du145, LNCap and VCap). The subcellular localization profiles were compared and analyzed by Chipview, the program for image database management and analysis. Results: A protein, GRK2 was identified to have unique localization in some of the cancer cells: localized in the membrane of two cancer cell lines, and in the cytosol of normal cell lines and one cancer cell line. The identified subcellular localization difference was confirmed by individual immunostaining. Conclusion: DAMA staining technology could be a powerful method for global subcellular localization profiling in high throughput format.