Development of Microscale Separation and Blotting Methods for Analysis of Small-Volume Biological Mixtures.

Capillary Electrophoresis;Western Blotting;Immunoblot;Chemistry;Science;Chemistry

Anderson, Gwendolyn JudithTakayama, Shuichi ;

University of Michigan

关键词: Capillary Electrophoresis;    Western Blotting;    Immunoblot;    Chemistry;    Science;    Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/96173/gjanders_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

A microscale Western blotting system based on separating sodium-dodecyl sulfate protein complexes by capillary gel electrophoresis followed by deposition onto a blotting membrane for immunoassay is described. In the system, the separation capillary is grounded through a sheath capillary to a mobile X-Y translation stage, which moves a blotting membrane past the capillary outlet for protein deposition. The blotting membrane is moistened with a methanol and buffer mixture to facilitate protein adsorption. In the first instrument discrete protein zones could be detected, but bands were broadened by about 1.7-fold by transfer to membrane. A complete Western blot for lysozyme was completed in about one hour with 50 pg mass detection limit from low microgram per milliliter samples. To increase throughput, a multiplexed CE-Western apparatus was constructed. To improve stability and allow for speedier separation, formulations for dextran sieving media were tested. Reduced band broadening and separation of 100 kDa proteins in 20 min was achieved. Up to four capillaries were operated in parallel, separating and transferring cell lysate proteins to a single blot for immunoassay for total analysis in 1 hour. Each capillary performed multiple injections before capillary regeneration, further increasing throughput. Sub-microgram/mL levels of actin were easily detected in cell lysate samples. These results demonstrate substantial reduction in time requirements and improvement in mass sensitivity compared to conventional Western blots. Dot blotting using capillaries for deposition is also reported. By delivering nanoliter-microliter volumes to a narrow spot, sensitivity is enhanced. For a dot 1000x larger in volume, a 7x larger diameter dot was observed, representing orders of magnitude in concentrating effects for larger volume accumulations. Western blotting using capillary electrophoresis and capillary dot blotting show promise to analyze low volume samples with reduced reagents and time. These methods retain the information content of a typical immunoblot and extend analytical capabilities for both Western and dot blotting.