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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

Increased local concentrations of protein bands upon pulsing. Protein band intensity analyses in FIGE (a I), CFE (a II), and CFE followed by resting within glass plates in room temperature for 12 hours (a III). Lanes 1 to 6 are 2 μL, 4 μL, 6 μL, 8 μL, 10 μL, and 12 μL of Mark12 protein standards, respectively, in a self-cast Bio-Rad 14% SDS-PAGE 1 mm × 7 cm gel followed by Coomassie blue staining. a I) Gel was run with a pulsed-field at (4 sec/3.4 sec) at 200 V for 13 hours, with an average buffer temperature of 30°C. A II) Gel was run at a constant field of 200 V for one hour and an average buffer temperature of 25°C. a III) Gel was run at a constant field of 200 V for one hour and left at rest for another 12 hours within the glass plates to permit diffusion prior to staining. b) Densitometry analysis of protein bands in the gels of the three conditions tested. Molecular mass was represented by alphabet A to K, where A = 200 kDa, B = 116.3 kDa, C = 97.4 kDa, D = 66.3 kDa, E = 55.4 kDa, F = 36.3 kDa, G = 31.0 kDa, H = 21.5 kDa, I = 14.4 kDa, J = 6.0 kDa, and K = unresolved 3.5/2.0 kDa bands, respectively. Migration distance relative to the dye front (Rf) and intensity of bands from lane 6 of all three gels was densitometrically analyzed using Quantity One software. The graph results were the average of two independent experiments. The graph results were subsequently employed in the calculation of peak variance, σ2, in Table 1.
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Figure 2: Increased local concentrations of protein bands upon pulsing. Protein band intensity analyses in FIGE (a I), CFE (a II), and CFE followed by resting within glass plates in room temperature for 12 hours (a III). Lanes 1 to 6 are 2 μL, 4 μL, 6 μL, 8 μL, 10 μL, and 12 μL of Mark12 protein standards, respectively, in a self-cast Bio-Rad 14% SDS-PAGE 1 mm × 7 cm gel followed by Coomassie blue staining. a I) Gel was run with a pulsed-field at (4 sec/3.4 sec) at 200 V for 13 hours, with an average buffer temperature of 30°C. A II) Gel was run at a constant field of 200 V for one hour and an average buffer temperature of 25°C. a III) Gel was run at a constant field of 200 V for one hour and left at rest for another 12 hours within the glass plates to permit diffusion prior to staining. b) Densitometry analysis of protein bands in the gels of the three conditions tested. Molecular mass was represented by alphabet A to K, where A = 200 kDa, B = 116.3 kDa, C = 97.4 kDa, D = 66.3 kDa, E = 55.4 kDa, F = 36.3 kDa, G = 31.0 kDa, H = 21.5 kDa, I = 14.4 kDa, J = 6.0 kDa, and K = unresolved 3.5/2.0 kDa bands, respectively. Migration distance relative to the dye front (Rf) and intensity of bands from lane 6 of all three gels was densitometrically analyzed using Quantity One software. The graph results were the average of two independent experiments. The graph results were subsequently employed in the calculation of peak variance, σ2, in Table 1.

Mentions: We investigated the effect of pulsing on the protein band intensity across a range of molecular masses; twelve protein species were separated by 14 % PAGE. The results showed that FIGE increased band intensities (Figure 2a I) compared with the CFE control (Figure 2a II). This notion was further investigated quantitatively using densitometry (Figure 2b). Protein peak heights were increased two-fold for protein molecular mass lower than 66 kDa. The enhancement of intensity for protein with molecular mass larger than 97 kDa was not obvious at this gel percentage and under these pulsing conditions. The increase in band intensities may be a result of reduced band diffusion and trapping of protein molecules in the gel matrix during migration. Diffusion became apparent when a control gel was run and allowed to rest for a further 12 hours within the glass plates at 20°C prior to staining (see Figure 2a III). The differences in band intensities were not caused by artifacts in staining and scanning as the parameters for staining and scanned were identical within the same set of experiments. Diffusion was not due to an increase in temperature as the temperature of gel during pulsing was 5°C higher than that of constant field controls. It was apparent that FIGE reduced band diffusion over a longer run time, since the run time required in this case was approximately 13-fold greater than that of the control. A general reduction in the full-width-half-maximum measure as a result of pulsing with respect to control was observed, suggesting that pulsing improved the overall efficiency of protein separation (Table 1). The improvement in separation efficiency becomes less obvious for relatively high molecular mass (≥ 200 kDa) and relatively low molecular mass (≤ 21.5 kDa) proteins. In summary, protein band intensity could be enhanced using FIGE.


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)

Increased local concentrations of protein bands upon pulsing. Protein band intensity analyses in FIGE (a I), CFE (a II), and CFE followed by resting within glass plates in room temperature for 12 hours (a III). Lanes 1 to 6 are 2 μL, 4 μL, 6 μL, 8 μL, 10 μL, and 12 μL of Mark12 protein standards, respectively, in a self-cast Bio-Rad 14% SDS-PAGE 1 mm × 7 cm gel followed by Coomassie blue staining. a I) Gel was run with a pulsed-field at (4 sec/3.4 sec) at 200 V for 13 hours, with an average buffer temperature of 30°C. A II) Gel was run at a constant field of 200 V for one hour and an average buffer temperature of 25°C. a III) Gel was run at a constant field of 200 V for one hour and left at rest for another 12 hours within the glass plates to permit diffusion prior to staining. b) Densitometry analysis of protein bands in the gels of the three conditions tested. Molecular mass was represented by alphabet A to K, where A = 200 kDa, B = 116.3 kDa, C = 97.4 kDa, D = 66.3 kDa, E = 55.4 kDa, F = 36.3 kDa, G = 31.0 kDa, H = 21.5 kDa, I = 14.4 kDa, J = 6.0 kDa, and K = unresolved 3.5/2.0 kDa bands, respectively. Migration distance relative to the dye front (Rf) and intensity of bands from lane 6 of all three gels was densitometrically analyzed using Quantity One software. The graph results were the average of two independent experiments. The graph results were subsequently employed in the calculation of peak variance, σ2, in Table 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Increased local concentrations of protein bands upon pulsing. Protein band intensity analyses in FIGE (a I), CFE (a II), and CFE followed by resting within glass plates in room temperature for 12 hours (a III). Lanes 1 to 6 are 2 μL, 4 μL, 6 μL, 8 μL, 10 μL, and 12 μL of Mark12 protein standards, respectively, in a self-cast Bio-Rad 14% SDS-PAGE 1 mm × 7 cm gel followed by Coomassie blue staining. a I) Gel was run with a pulsed-field at (4 sec/3.4 sec) at 200 V for 13 hours, with an average buffer temperature of 30°C. A II) Gel was run at a constant field of 200 V for one hour and an average buffer temperature of 25°C. a III) Gel was run at a constant field of 200 V for one hour and left at rest for another 12 hours within the glass plates to permit diffusion prior to staining. b) Densitometry analysis of protein bands in the gels of the three conditions tested. Molecular mass was represented by alphabet A to K, where A = 200 kDa, B = 116.3 kDa, C = 97.4 kDa, D = 66.3 kDa, E = 55.4 kDa, F = 36.3 kDa, G = 31.0 kDa, H = 21.5 kDa, I = 14.4 kDa, J = 6.0 kDa, and K = unresolved 3.5/2.0 kDa bands, respectively. Migration distance relative to the dye front (Rf) and intensity of bands from lane 6 of all three gels was densitometrically analyzed using Quantity One software. The graph results were the average of two independent experiments. The graph results were subsequently employed in the calculation of peak variance, σ2, in Table 1.
Mentions: We investigated the effect of pulsing on the protein band intensity across a range of molecular masses; twelve protein species were separated by 14 % PAGE. The results showed that FIGE increased band intensities (Figure 2a I) compared with the CFE control (Figure 2a II). This notion was further investigated quantitatively using densitometry (Figure 2b). Protein peak heights were increased two-fold for protein molecular mass lower than 66 kDa. The enhancement of intensity for protein with molecular mass larger than 97 kDa was not obvious at this gel percentage and under these pulsing conditions. The increase in band intensities may be a result of reduced band diffusion and trapping of protein molecules in the gel matrix during migration. Diffusion became apparent when a control gel was run and allowed to rest for a further 12 hours within the glass plates at 20°C prior to staining (see Figure 2a III). The differences in band intensities were not caused by artifacts in staining and scanning as the parameters for staining and scanned were identical within the same set of experiments. Diffusion was not due to an increase in temperature as the temperature of gel during pulsing was 5°C higher than that of constant field controls. It was apparent that FIGE reduced band diffusion over a longer run time, since the run time required in this case was approximately 13-fold greater than that of the control. A general reduction in the full-width-half-maximum measure as a result of pulsing with respect to control was observed, suggesting that pulsing improved the overall efficiency of protein separation (Table 1). The improvement in separation efficiency becomes less obvious for relatively high molecular mass (≥ 200 kDa) and relatively low molecular mass (≤ 21.5 kDa) proteins. In summary, protein band intensity could be enhanced using FIGE.

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