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Increase in local protein concentration by field-inversion gel electrophoresis.

Tsai H, Low TY, Freeby S, Paulus A, Ramnarayanan K, Cheng CP, Leung HC - Proteome Sci (2007)

Bottom Line: 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.Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE.

View Article: PubMed Central - HTML - PubMed

Affiliation: Medical Proteomics and Bioanalysis Section, Genome Institute of Singapore, Singapore. tsaihh@gis.a-star.edu.sg.

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.

No MeSH data available.


Related in: MedlinePlus

Electric circuit of pulse generator and field diagram. a) Electric circuit diagram for generating positive (+E) and negative (-E) square-wave electric fields during field inversion experiments. Direct current (DC) supply is from an external source. NC, normally closed switch; AC relay, alternate current relay; Com, common outlet. Inset b) shows a profile of the electric field during a typical FIGE experiment where + E = - E and ta (forward field time) is longer than tr (reverse field time).
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Figure 1: Electric circuit of pulse generator and field diagram. a) Electric circuit diagram for generating positive (+E) and negative (-E) square-wave electric fields during field inversion experiments. Direct current (DC) supply is from an external source. NC, normally closed switch; AC relay, alternate current relay; Com, common outlet. Inset b) shows a profile of the electric field during a typical FIGE experiment where + E = - E and ta (forward field time) is longer than tr (reverse field time).

Mentions: We report here the engineering of a simple field-inversion device (Figure 1) and an initial study of protein separation efficiency using FIGE. The focus is to determine the ability of FIGE in the reduction of diffusion of a wide spectrum of protein species in a PAG matrix. The reduction of protein diffusion in polyacrylamide gel electrophoresis may lead to higher protein concentration per unit gel volume. The increased local protein concentration may therefore enhance tryptic peptide recovery in subsequent mass spectrometry analysis.


Increase in local protein concentration by field-inversion gel electrophoresis.

Tsai H, Low TY, Freeby S, Paulus A, Ramnarayanan K, Cheng CP, Leung HC - Proteome Sci (2007)

Electric circuit of pulse generator and field diagram. a) Electric circuit diagram for generating positive (+E) and negative (-E) square-wave electric fields during field inversion experiments. Direct current (DC) supply is from an external source. NC, normally closed switch; AC relay, alternate current relay; Com, common outlet. Inset b) shows a profile of the electric field during a typical FIGE experiment where + E = - E and ta (forward field time) is longer than tr (reverse field time).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211458&req=5

Figure 1: Electric circuit of pulse generator and field diagram. a) Electric circuit diagram for generating positive (+E) and negative (-E) square-wave electric fields during field inversion experiments. Direct current (DC) supply is from an external source. NC, normally closed switch; AC relay, alternate current relay; Com, common outlet. Inset b) shows a profile of the electric field during a typical FIGE experiment where + E = - E and ta (forward field time) is longer than tr (reverse field time).
Mentions: We report here the engineering of a simple field-inversion device (Figure 1) and an initial study of protein separation efficiency using FIGE. The focus is to determine the ability of FIGE in the reduction of diffusion of a wide spectrum of protein species in a PAG matrix. The reduction of protein diffusion in polyacrylamide gel electrophoresis may lead to higher protein concentration per unit gel volume. The increased local protein concentration may therefore enhance tryptic peptide recovery in subsequent mass spectrometry analysis.

Bottom Line: 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.Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE.

View Article: PubMed Central - HTML - PubMed

Affiliation: Medical Proteomics and Bioanalysis Section, Genome Institute of Singapore, Singapore. tsaihh@gis.a-star.edu.sg.

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.

No MeSH data available.


Related in: MedlinePlus