Methodology

Increase in local protein concentration by field-inversion gel electrophoresis

Henghang Tsai¹ , Teck Yew Low¹ , Steve Freeby² , Aran Paulus² , Kalpana Ramnarayanan¹ , Chung-pui Paul Cheng¹ and Hon-chiu Eastwood Leung³

¹  Medical Proteomics and Bioanalysis Section, Genome Institute of Singapore, Singapore

²  Bio-Rad Laboratories, Hercules, USA

³  Department of Molecular and Cellular Biology, Department of Pediatrics, Baylor College of Medicine and Texas Children's Hospital, Houston, USA

author email corresponding author email

Proteome Science 2007, 5:18doi:10.1186/1477-5956-5-18

Published:	26 September 2007

Abstract

Background

Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE).

Results

Separation of model protein species and large protein complexes was compared between FIGE and constant field electrophoresis (CFE) in different percentages of PAGs. Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times. These results revealed an increase in band intensity per defined gel volume. A biphasic protein relative mobility shift was observed in percentages of PAGs up to 14%. However, the effect of FIGE on protein separation was stochastic at higher PAG percentage. Rat liver lysates subjected to FIGE in the second-dimension separation of two-dimensional polyarcylamide gel electrophoresis (2D PAGE) showed a 20% increase in the number of discernible spots compared with CFE. Nine common spots from both FIGE and CFE were selected for peptide sequencing by mass spectrometry (MS), which revealed higher final ion scores of all nine protein spots from FIGE. Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE.

Conclusion

The present investigation suggests that FIGE under appropriate conditions improves protein separation efficiency during PAGE as a result of increased local protein concentration. FIGE can be implemented with minimal additional instrumentation in any laboratory setting. Despite the tradeoff of longer running times, FIGE can be a powerful protein separation tool.