Microfluidic integration of Western blotting is enabled by electrotransfer-assisted sodium dodecyl sulfate dilution.
Analyst
; 138(1): 158-63, 2013 Jan 07.
Article
en En
| MEDLINE
| ID: mdl-23042290
ABSTRACT
We integrate sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with subsequent antibody probing in a single, monolithic microdevice to realize microfluidic Western blotting. A hurdle to successful on-chip Western blotting lies in restoring antibody recognition of previously sized (denatured, reduced) proteins. To surmount this hurdle, we locally dilute free SDS from SDS-protein complexes using differential electromigration of the species during electrotransfer between SDS-PAGE and blotting regions of a microchamber. Local dilution of SDS minimizes re-association of SDS with proteins offering means to restore antibody binding affinity to proteins after SDS-PAGE. To achieve automated, programmable operation in a single instrument, we utilize a 1 × 2 mm(2) glass microchamber photopatterned with spatially distinct, contiguous polyacrylamide regions for SDS-PAGE, electrotransfer, and antibody blotting. Optimization of both the SDS-PAGE and electrotransfer conditions yields transfer distances of <1 mm (40 s). The Western blot is completed in 180 s, with fully automated assay operation using programmable voltage control. After SDS-PAGE and electrotransfer, we observe ~80% capture of protein band mass on the blotting region for a model protein, C-reactive protein. This novel microfluidic Western blot approach introduces fine transport control for in-transit protein handling to form the basis for an automated, rapid alternative to conventional slab-gel Western blotting.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Dodecil Sulfato de Sodio
/
Western Blotting
/
Técnicas Analíticas Microfluídicas
Límite:
Animals
Idioma:
En
Revista:
Analyst
Año:
2013
Tipo del documento:
Article
País de afiliación:
Estados Unidos