学位论文详细信息
Development of Microfluidic Based Western Blot Technology for Fast and High-Content Analyses.
Microfluidic based Western blot;Chemistry;Science;Chemistry
Jin, ShiMapp, Anna K ;
University of Michigan
关键词: Microfluidic based Western blot;    Chemistry;    Science;    Chemistry;   
Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/116663/shjin_1.pdf?sequence=1&isAllowed=y
瑞士|英语
来源: The Illinois Digital Environment for Access to Learning and Scholarship
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【 摘 要 】

Western blotting has been a highly valued method in research for protein identification and relative quantitation in complex biological samples. Although widely adapted, Western blot assays have many limitations including labor intensive, low throughput, low information content, large sample consumption, and mediocre repeatability.A microfluidic Western blotting system based on protein sieving on microchips followed by direct blotting onto a moving membrane is described. By flowing same separation sieving buffer by the sides of separation channel, protein bands are well defined and transferred onto membrane with minimum band broadening, which is an improvement to a previous hybrid capillary Western blotting system. Separation speed is significantly improved on a microchip due to a high electric field applied. A complete Western blot for actin is finished in about 25 min with 0.7 nM detection limit.While speed of separation is important to some applications, in many cases resolution between closely related protein isoforms is required. 2 kDa different proteins are used to demonstrate the achievement of improved separation resolution by modifying channel dimensions and lengths. With an 8 cm long separation channel, ERK1 (44 kDa) and ERK2 (42 kDa) are baseline resolved on the membrane within 8 min.Another advantage of using microfluidic devices for Western blotting is the potential for high throughput and high content analysis. Up to seven parallel separation channels, each with 3 sample reservoirs, are accommodated on a 3 inch by 3 inch glass chip. 21 Western blots are completed within 30 min. To demonstrate the capability of detecting multiple proteins using a small amount of cell lysate sample, 11 proteins are detected using less than 200 nL lysate sample (0.4 μg total protein). For comparison, traditional assays would require at least 90 μL (90 μg total protein) to finish the same task.Overall, microfluidic based Western blotting technology has shown promise for fast analysis, low sample consumption, and multiplexing over traditional methods.

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