A multi-channel gel electrophoresis and continuous fraction collection apparatus for high throughput protein separation and characterization
To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A Counter Free-Flow elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this system using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of 10-150 kDa; sample recovery rates were 50percent or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 L/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 g per channel and reduced resolution.
- Research Organization:
- Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Sponsoring Organization:
- Engineering Division; Genomics Division
- DOE Contract Number:
- DE-AC02-05CH11231
- OSTI ID:
- 983162
- Report Number(s):
- LBNL-3280E; TRN: US201014%%802
- Journal Information:
- Electrophoresis, Vol. 31, Issue 3; Related Information: Journal Publication Date: January 2010
- Country of Publication:
- United States
- Language:
- English
Similar Records
Colloid molecular weight estimation by gel chromatography/acrylamide gel electrophoresis
On-line detection of proteins in gel electrophoresis by ultraviolet absorption and by native fluorescence utilizing a charge-coupled device imaging system