Dataset Information

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

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.

SUBMITTER: Tsai H

PROVIDER: S-EPMC2211458 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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Publications

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

Tsai Henghang H Low Teck Yew TY Freeby Steve S Paulus Aran A Ramnarayanan Kalpana K Cheng Chung-Pui Paul CP Leung Hon-Chiu Eastwood HC

Proteome science 20070926

<h4>Background</h4>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-inversi ...[more]

PMID: 17897441

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Project description:Pulsed-field gel electrophoresis (PFGE) characterization of 335 temporally and spatially matched clinical, bovine, and human Salmonella enterica subsp. enterica isolates revealed 167 XbaI PFGE patterns. These isolates were previously classified into 51 serotypes and 73 sequence types, as determined by multilocus sequence typing. Discriminatory power of PFGE (Simpson's index, D = 0.991) was considerably higher than that of multilocus sequence typing (D = 0.920) or serotyping (D = 0.913). Although 128 PFGE types each only represented a single isolate, 8 PFGE types represented >4 isolates, including (i) three serotype Enteritidis and Heidelberg patterns that were only identified among human isolates, (ii) two PFGE patterns (each representing serotypes Bardo and Newport) that were significantly more common among bovine isolates as compared with human isolates; (iii) two PFGE types that each includes two serotypes (4,5,12:i:- and Typhimurium; Thompson and 1,7:-:1,5); and (iv) one PFGE type that includes eight Typhimurium isolates from humans and cattle. Characterization of isolates collected over multiple farm visits indicated that given specific PFGE types persisted over time on 11 farms. On an additional seven farms, isolates with a given sequence type represented multiple PFGE type, which typically only differed by <3 bands, suggesting PFGE type diversification during strain persistence. Sixteen PFGE types were isolated from 2 or more farms, including two widely distributed serotype Newport-associated PFGE types each found on 10 farms. In six instances two or three human isolates collected in the same county in the same or consecutive months represented the same subtypes, suggesting small human case clusters. PFGE-based characterization and surveillance of human and animal isolates can provide improved understanding of Salmonella diversity and epidemiology, including identification of possible host-associated and common, widely distributed PFGE types.

Dataset Information

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

Background

Results

Conclusion

Publications

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

Similar Datasets

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