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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

Effects of FIGE on protein separation under native gel conditions. Comparison of GroEL 14-mer complex (840 kDa) (a) or MCF-7 cell nuclear extract (b) in native PAG between CFE and FIGE conditions. FIGE was the left panel. CFE control was the right panel. The gels were 1 mm × 7 cm native 6% PAG casted in a mini-Protean 3 apparatus. Run time was 2 hrs 15 min in control condition. Pulsed condition run time was 5 hrs 30 min with a ta/tr of 400/100 msec. Lane 1, native MW markers (10 μg total); Lane 2, 10 μg purified E. coli GroEL native complex (14-mer, 840 kDa). The band at 300 kDa could be a minor cofactor associated with GroEL during purification. The gel was stained with Coomassie blue. Proteins were purposefully overloaded to best represent the effect of "detrapping" of native proteins under pulsing conditions. In panel b), MCF-7 nuclear extracted were run through native 2–5% PAG. The run time was 10 hrs for CFE control condition. Pulsed condition run time was 14 hrs with ta/tr of 900 msec/240 msec. Lane 1, 5.6 μg MCF-7 cell nuclear extract; Lane 2, 7.5 μg MCF-7 cell nuclear extract; Lane 3, native protein MW markers. The gels were silver stained.
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Figure 5: Effects of FIGE on protein separation under native gel conditions. Comparison of GroEL 14-mer complex (840 kDa) (a) or MCF-7 cell nuclear extract (b) in native PAG between CFE and FIGE conditions. FIGE was the left panel. CFE control was the right panel. The gels were 1 mm × 7 cm native 6% PAG casted in a mini-Protean 3 apparatus. Run time was 2 hrs 15 min in control condition. Pulsed condition run time was 5 hrs 30 min with a ta/tr of 400/100 msec. Lane 1, native MW markers (10 μg total); Lane 2, 10 μg purified E. coli GroEL native complex (14-mer, 840 kDa). The band at 300 kDa could be a minor cofactor associated with GroEL during purification. The gel was stained with Coomassie blue. Proteins were purposefully overloaded to best represent the effect of "detrapping" of native proteins under pulsing conditions. In panel b), MCF-7 nuclear extracted were run through native 2–5% PAG. The run time was 10 hrs for CFE control condition. Pulsed condition run time was 14 hrs with ta/tr of 900 msec/240 msec. Lane 1, 5.6 μg MCF-7 cell nuclear extract; Lane 2, 7.5 μg MCF-7 cell nuclear extract; Lane 3, native protein MW markers. The gels were silver stained.

Mentions: We further explored the application of FIGE in the separation of protein complexes in native gel conditions. We discovered that the purified E. coli GroEL 14-mer complex (840 kDa) could effectively condense and migrate into the gel matrix under pulsing conditions of 900/240 msec (Figure 5a right panel). In contrast, the GroEL 14-mer complex migrated in a stochastic fashion under constant field conditions (see Figure 5a left panel). The difference was not due to an inappropriate buffer composition as the appearance of the GroEL 14-mer complex band was sharper than that in the CFE control. In addition, the protein bands of the MW protein markers were sharper after pulsing than the diffused bands in the control (see Figure 5a). Next, we tested a complex sample using MCF-7 cell line nuclear extract under native PAGE conditions. The results revealed that pulsing effectively condensed protein complexes greater than 2000 kDa in size, with a better resolution and band intensity (Figure 5b). In summary, FIGE might also be used to resolve very high molecular mass (MW > 1000 kDa) protein complexes by native PAGE.


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)

Effects of FIGE on protein separation under native gel conditions. Comparison of GroEL 14-mer complex (840 kDa) (a) or MCF-7 cell nuclear extract (b) in native PAG between CFE and FIGE conditions. FIGE was the left panel. CFE control was the right panel. The gels were 1 mm × 7 cm native 6% PAG casted in a mini-Protean 3 apparatus. Run time was 2 hrs 15 min in control condition. Pulsed condition run time was 5 hrs 30 min with a ta/tr of 400/100 msec. Lane 1, native MW markers (10 μg total); Lane 2, 10 μg purified E. coli GroEL native complex (14-mer, 840 kDa). The band at 300 kDa could be a minor cofactor associated with GroEL during purification. The gel was stained with Coomassie blue. Proteins were purposefully overloaded to best represent the effect of "detrapping" of native proteins under pulsing conditions. In panel b), MCF-7 nuclear extracted were run through native 2–5% PAG. The run time was 10 hrs for CFE control condition. Pulsed condition run time was 14 hrs with ta/tr of 900 msec/240 msec. Lane 1, 5.6 μg MCF-7 cell nuclear extract; Lane 2, 7.5 μg MCF-7 cell nuclear extract; Lane 3, native protein MW markers. The gels were silver stained.
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Figure 5: Effects of FIGE on protein separation under native gel conditions. Comparison of GroEL 14-mer complex (840 kDa) (a) or MCF-7 cell nuclear extract (b) in native PAG between CFE and FIGE conditions. FIGE was the left panel. CFE control was the right panel. The gels were 1 mm × 7 cm native 6% PAG casted in a mini-Protean 3 apparatus. Run time was 2 hrs 15 min in control condition. Pulsed condition run time was 5 hrs 30 min with a ta/tr of 400/100 msec. Lane 1, native MW markers (10 μg total); Lane 2, 10 μg purified E. coli GroEL native complex (14-mer, 840 kDa). The band at 300 kDa could be a minor cofactor associated with GroEL during purification. The gel was stained with Coomassie blue. Proteins were purposefully overloaded to best represent the effect of "detrapping" of native proteins under pulsing conditions. In panel b), MCF-7 nuclear extracted were run through native 2–5% PAG. The run time was 10 hrs for CFE control condition. Pulsed condition run time was 14 hrs with ta/tr of 900 msec/240 msec. Lane 1, 5.6 μg MCF-7 cell nuclear extract; Lane 2, 7.5 μg MCF-7 cell nuclear extract; Lane 3, native protein MW markers. The gels were silver stained.
Mentions: We further explored the application of FIGE in the separation of protein complexes in native gel conditions. We discovered that the purified E. coli GroEL 14-mer complex (840 kDa) could effectively condense and migrate into the gel matrix under pulsing conditions of 900/240 msec (Figure 5a right panel). In contrast, the GroEL 14-mer complex migrated in a stochastic fashion under constant field conditions (see Figure 5a left panel). The difference was not due to an inappropriate buffer composition as the appearance of the GroEL 14-mer complex band was sharper than that in the CFE control. In addition, the protein bands of the MW protein markers were sharper after pulsing than the diffused bands in the control (see Figure 5a). Next, we tested a complex sample using MCF-7 cell line nuclear extract under native PAGE conditions. The results revealed that pulsing effectively condensed protein complexes greater than 2000 kDa in size, with a better resolution and band intensity (Figure 5b). In summary, FIGE might also be used to resolve very high molecular mass (MW > 1000 kDa) protein complexes by native PAGE.

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