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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.


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Changes in relative mobility upon different pulsing conditions. Comparison of changes in protein relative mobility between FIGE and CFE conditions in 6% (a), 10% (b), 14% (c) and 18% (d) cross-linked polyacrylamide concentration self-cast Bio-Rad SDS-PAG (1 mm × 7 cm). Different concentrations of polyacrylamide were casted in a mini-Protean 3 apparatus. Five microliters of Mark12 protein standards were used. Relative mobility was measured as a ratio of the migration distance of the target protein to that of the resolving front (% Rf). The graphs were generated using Quantity One software. The y-axis denotes the percent differences of % Rf in pulsed conditions compared to the CFE control. Each data point was the average of two separate experiments. All gels were run at 200 V with the average buffer temperature of 10°C. Positive values denote shorter migration distance and negative values denote longer migration distance with respect to CFE control. Error bar denotes the standard deviation of two separate experiments. Error bar cannot be showed if the range is smaller than the label.
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Figure 3: Changes in relative mobility upon different pulsing conditions. Comparison of changes in protein relative mobility between FIGE and CFE conditions in 6% (a), 10% (b), 14% (c) and 18% (d) cross-linked polyacrylamide concentration self-cast Bio-Rad SDS-PAG (1 mm × 7 cm). Different concentrations of polyacrylamide were casted in a mini-Protean 3 apparatus. Five microliters of Mark12 protein standards were used. Relative mobility was measured as a ratio of the migration distance of the target protein to that of the resolving front (% Rf). The graphs were generated using Quantity One software. The y-axis denotes the percent differences of % Rf in pulsed conditions compared to the CFE control. Each data point was the average of two separate experiments. All gels were run at 200 V with the average buffer temperature of 10°C. Positive values denote shorter migration distance and negative values denote longer migration distance with respect to CFE control. Error bar denotes the standard deviation of two separate experiments. Error bar cannot be showed if the range is smaller than the label.

Mentions: We studied the effects of different ratios of forward pulse times (ta) and backward pulse times (tr) on protein relative mobility under different percentages of PAGs. Relative mobility was defined as the ratio of the migration distance of the target protein to the migration distance of the dye front (Rf). The relative mobility was abbreviated as the percentage of Rf (% Rf). The % Rf did not involve run time as it measured the position of the proteins when the dye front reached the same position regardless of the run time. Biphasic changes of % Rf were observed at 6%, 10%, and 14% of PAGs (Figure 3a to 3c) under the tested pulsing conditions. More reduction in % Rf was observed with protein size of 36 kDa to 66 kDa. The reduction decreased when protein species were more massive than 66 kDa. The apex (i.e., the molecular mass that showed the maximum difference in % Rf) shifted to smaller molecular mass species when the gel percentage increased (Figure 3a, 3b, and 3c). Higher frequencies pulse cycles (ta/tr = 60/16 msec, 150/40 msec, and 300/80 msec) resulted in more obvious differences in relative mobility compared to low frequency pulse cycle (ta/tr = 900/240 msec) (Figure 3a to 3b). This change upon different pulse cycles was observed in the repeat run (Figure 3a and 3b). However, we did not see a simple linear relationship changes in relative mobility to gel concentration, pulse frequency, and protein size. We observed negative values in differences of % Rf (Figure 3c and 3d) relative to CFE at high percentages of PAGs, suggesting that the target proteins revealed a longer migration distance. However, we did not observe a consistent pattern of altered relative mobility at 18 % PAG. The migration of proteins in such a high polyacrylamide concentration was apparently a stochastic process (Figure 3d). In summary, the results showed that the maximum difference in relative mobility was not a simple linear relationship with molecular mass, pulse cycles, and gel percentage.


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)

Changes in relative mobility upon different pulsing conditions. Comparison of changes in protein relative mobility between FIGE and CFE conditions in 6% (a), 10% (b), 14% (c) and 18% (d) cross-linked polyacrylamide concentration self-cast Bio-Rad SDS-PAG (1 mm × 7 cm). Different concentrations of polyacrylamide were casted in a mini-Protean 3 apparatus. Five microliters of Mark12 protein standards were used. Relative mobility was measured as a ratio of the migration distance of the target protein to that of the resolving front (% Rf). The graphs were generated using Quantity One software. The y-axis denotes the percent differences of % Rf in pulsed conditions compared to the CFE control. Each data point was the average of two separate experiments. All gels were run at 200 V with the average buffer temperature of 10°C. Positive values denote shorter migration distance and negative values denote longer migration distance with respect to CFE control. Error bar denotes the standard deviation of two separate experiments. Error bar cannot be showed if the range is smaller than the label.
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Figure 3: Changes in relative mobility upon different pulsing conditions. Comparison of changes in protein relative mobility between FIGE and CFE conditions in 6% (a), 10% (b), 14% (c) and 18% (d) cross-linked polyacrylamide concentration self-cast Bio-Rad SDS-PAG (1 mm × 7 cm). Different concentrations of polyacrylamide were casted in a mini-Protean 3 apparatus. Five microliters of Mark12 protein standards were used. Relative mobility was measured as a ratio of the migration distance of the target protein to that of the resolving front (% Rf). The graphs were generated using Quantity One software. The y-axis denotes the percent differences of % Rf in pulsed conditions compared to the CFE control. Each data point was the average of two separate experiments. All gels were run at 200 V with the average buffer temperature of 10°C. Positive values denote shorter migration distance and negative values denote longer migration distance with respect to CFE control. Error bar denotes the standard deviation of two separate experiments. Error bar cannot be showed if the range is smaller than the label.
Mentions: We studied the effects of different ratios of forward pulse times (ta) and backward pulse times (tr) on protein relative mobility under different percentages of PAGs. Relative mobility was defined as the ratio of the migration distance of the target protein to the migration distance of the dye front (Rf). The relative mobility was abbreviated as the percentage of Rf (% Rf). The % Rf did not involve run time as it measured the position of the proteins when the dye front reached the same position regardless of the run time. Biphasic changes of % Rf were observed at 6%, 10%, and 14% of PAGs (Figure 3a to 3c) under the tested pulsing conditions. More reduction in % Rf was observed with protein size of 36 kDa to 66 kDa. The reduction decreased when protein species were more massive than 66 kDa. The apex (i.e., the molecular mass that showed the maximum difference in % Rf) shifted to smaller molecular mass species when the gel percentage increased (Figure 3a, 3b, and 3c). Higher frequencies pulse cycles (ta/tr = 60/16 msec, 150/40 msec, and 300/80 msec) resulted in more obvious differences in relative mobility compared to low frequency pulse cycle (ta/tr = 900/240 msec) (Figure 3a to 3b). This change upon different pulse cycles was observed in the repeat run (Figure 3a and 3b). However, we did not see a simple linear relationship changes in relative mobility to gel concentration, pulse frequency, and protein size. We observed negative values in differences of % Rf (Figure 3c and 3d) relative to CFE at high percentages of PAGs, suggesting that the target proteins revealed a longer migration distance. However, we did not observe a consistent pattern of altered relative mobility at 18 % PAG. The migration of proteins in such a high polyacrylamide concentration was apparently a stochastic process (Figure 3d). In summary, the results showed that the maximum difference in relative mobility was not a simple linear relationship with molecular mass, pulse cycles, and gel percentage.

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