Determination of relative protein abundance by internally normalized ratio algorithm with antibody arrays.
In this paper, we report an experimental setup and mathematical algorithm for determination of relative protein abundance from directly labeled native protein samples applied to an array of antibodies. The application of the proposed experimental system compensates internally at each array element for a number of deficiencies in array experiments such as differential labeling efficiency in dual color assay systems, differential solubility of protein molecules in dual color assay systems, and differential affinity of capture reagents toward proteins labeled with two different fluorescent dyes. This system offers full compensation for variable amounts of capture reagents on separate array structures, as well as limited compensation for nonspecific interactions between capture reagents and analytes. The proposed experimental strategy enables the use of a large number of capture reagents to develop a true multiplex analysis system that will yield complete relative protein abundance information in two biological systems.
Andersson
Oskar
O
BD Biosciences Clontech, 1020 E. Meadow Circle, Palo Alto, California 94303, USA.
Kozlowski
Mark
M
Garachtchenko
Tatiana
T
Nikoloff
Corina
C
Lew
Nancy
N
Litman
David J
DJ
Chaga
Grigoriy
G
D000465:Algorithms; D000911:Antibodies, Monoclonal; D002457:Cell Extracts / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007118:Immunoassay; D007202:Indicators and Reagents; D019169:Jurkat Cells; D040081:Protein Array Analysis; D011506:Proteins / Q000032:analysis*
D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D007202:Indicators and Reagents; D011506:Proteins