Protein precipitation method for salivary proteins and rehydration buffer for two-dimensional electrophoresis

Jessie, K.; Hashim, O.H.; Rahim, Z.H.A. (2008) Protein precipitation method for salivary proteins and rehydration buffer for two-dimensional electrophoresis. Biotechnology, 7 (4). pp. 686-693. ISSN 1682-296X

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    Abstract

    Precipitants for salivary proteins and rehydration buffers for two-dimensional electrophoresis (2-DE) analysis were, respectively compared and evaluated. Five different protein precipitants: TCA, TCA-acetone-DTT, TCA-acetone-mercaptoethanol, acetone and alcohol were used to precipitate proteins of the saliva samples. The efficiency of the precipitants was evaluated from protein content of the precipitate reflecting protein recovery. The precipitate with the highest protein content was subsequently solubilized using different rehydration buffers (RB1, RB2, RB3 and RB4) before being subjected to the 2-DE. The efficiency of the different rehydration buffers was compared with respect to the resolution and focusing time taken to attain the maximum voltage. Each of the saliva samples was subjected to the above experiments, carried out in triplicates. The precipitant containing TCA-acetone-DTT exhibited the highest protein recovery (82.2) demonstrating significant difference when compared with the other precipitants (p<0.05). The RB4 containing DTT (reducing agent) and 0.5 IPG buffer 3-10 non-linear (carrier ampholyte) exhibited more protein spots indicating better separation resolution. The results obtained suggested that protein recovery depends on the precipitant used in the precipitation and resolution of proteins separation is influenced by the reducing agent and the ampholyte used in the rehydration buffer

    Item Type: Article
    Creators:
    1. Jessie, K.
    2. Hashim, O.H.(Department of Molecular Medicine, Faculty of Medicine Building, University of Malaya, 50603 Kuala Lumpur, MALAYSIA)
    3. Rahim, Z.H.A.
    Journal or Publication Title: Biotechnology
    Uncontrolled Keywords: Salivary proteins ; precipitants ; reducing agent ; carrier ampholytes
    Subjects: R Medicine
    Divisions: Faculty of Medicine
    Depositing User: Ms Haslinda Lahuddin
    Date Deposited: 10 Jul 2012 09:25
    Last Modified: 10 Jul 2012 09:25
    URI: http://eprints.um.edu.my/id/eprint/3437

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