Label-free detection of proteins in crude cell lysate with antibody arrays by a surface plasmon resonance imaging technique.
We established a label-free method of measuring proteins in crude cell lysate using antibody arrays and surface plasmon resonance (SPR) imaging. The refractivity of the running buffer was adjusted with that of the lysate to overcome the bulk effect. The chemistries of the fabricated arrays were investigated to reduce nonspecific adsorption on the array surface. We found that the hydrophilicity of the poly(ethylene glycol) moiety and lower electrostatic charge on the surface provided a specific measurement of antigen-antibody interaction. We validated the system by measuring the expression of eight proteins in the mouse brain and comparing the results to those by conventional Western blotting. The detection limit of the antibody array was approximately 30 ng/mL in crude cell lysate, on the same order as that of previous SPR research. The system enabled quick, label-free, and high-throughput analysis of abundant proteins with minimal sample volume ( approximately 200 muL). It is expected that our SPR antibody array will be applicable for direct protein expression profiling of cell lysate, as well as for cell phenotyping, food analysis, discovery of new biomarkers, and immunological disease diagnostics.
Kyo
Motoki
M
Biotechnology Frontier Project, Toyobo Co., Ltd., Tsuruga, Fukui, Japan. Motoki_Kyo@bio.toyobo.co.jp
Usui-Aoki
Kazue
K
Koga
Hisashi
H
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D002457:Cell Extracts / Q000737:chemistry*; D008297:Male; D051379:Mice; D015394:Molecular Structure; D011092:Polyethylene Glycols; D040081:Protein Array Analysis / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000276:immunology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance / Q000379:methods*
D000906:Antibodies; D002457:Cell Extracts; D011506:Proteins; D011092:Polyethylene Glycols