Increase in local protein concentration by field-inversion gel electrophoresis

Henghang Tsai,Kalpana Ramnarayanan,Hon-Chiu Leung,Aran Paulus,Steve Freeby,Teck Low,Chung-Pui Cheng

doi:10.1186/1477-5956-5-18

Abstract

BackgroundProteins 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).ResultsSeparation 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.ConclusionThe 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.

Highlights

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
The intensities of protein bands increase upon pulsing We investigated the effect of pulsing on the protein band intensity across a range of molecular masses; twelve protein species were separated by 14 % two-dimensional polyacrylamide gel electrophoresis (PAGE)
The results showed that field-inversion gel electrophoresis (FIGE) increased band intensities (Figure 2a I) compared with the constant field electrophoresis (CFE) control (Figure 2a II)

Summary

Introduction

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, decrease protein separation efficiency. Among the various pulsed-field gel electrophoresis techniques, FIGE is likely the easiest to perform with minimal special equipment that generates a highly uniform electric field across the gel [4] Both methods have been optimized to maximize efficiency (band width in the dimension of separation) and selectivity (distance between the center of two bands) for large DNA molecules. Published studies of the use of FIGE for protein separation are still lacking

Methods

Results

Discussion

Conclusion