The effect of cultivation conditions on protein composition of membrane of Paracoccus denitrificans
| Title in English | The effect of cultivation conditions on protein composition of Paracoccus denitrificans membrane |
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| Authors | |
| Year of publication | 2002 |
| Type | Article in Proceedings |
| Conference | VI. Pracovní setkání biochemiků a molekulárních biologů. Sborník příspěvků. |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | Paracoccus denitrificans; membrane proteins; proteome; 2-D electrophoresis |
| Description | New method using two-dimensional gel electrophoresis was optimised for Paracoccus denitrificans cell envelope study. This approach consist of sample preparation especially optimised for Paracoccus denitrificans membrane proteins, carrier-ampholyte isoelectric focusation in the first dimension (CA-IEF) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. Acquired 2-D gels were compared using image analysis and PDQUEST software. Various conditions of initial solubilisation using various detergents were tested. Sodium dodecyl sulphate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfo-nate (CHAPS), dodecyl-â-D-maltoside and 6-aminocaproic acid were used for this purpose. Sample buffer and IEF gel composition effects on 2-D gels resolution were also tested. Extraction using free detergent was compared with extraction using diluted detergent in mixture with sample buffer. Dilution ratio between presolubilised extract and sample buffer was optimised. Focusation algorithm was also optimised for this purpose. |
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