Direct determination of selenoproteins in polyvinylidene difluoride membranes by electrothermal atomic absorption spectrometry |
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Authors: | U Sidenius B Gammelgaard |
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Institution: | Department of Analytical and Pharmaceutical Chemistry, The Royal Danish School of Pharmacy, Universitetsparken 2, 2100 Copenhagen ?, Denmark e-mail: ulsi@mail.dfh.dk, DK
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Abstract: | A method for the direct determination of selenoproteins in plastic membranes after protein separation by gel electrophoresis
was developed. Quantification was based on the determination of the selenium content of the proteins by electrothermal atomic
absorption spectrometry (ET-AAS) after manual introduction of membrane pieces into the graphite furnace. The proteins were
separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene
difluoride (PVDF) membrane by semi-dry electroblotting. After staining the membrane, the protein bands were excised and chemical
modifier was added on top of the excised membrane prior to atomic absorption measurement. Acceptable linearity was achieved
in the range 2– 10 ng Se, corresponding to selenium concentrations close to 1 mg/L, when aqueous solutions of selenomethionine
standard as well as selenoprotein standard were applied to the membrane. A characteristic mass of 54 ± 4 pg/0.0044 s was obtained
for the selenoprotein standard. Protein transfer from polyacrylamide gel to the membrane was quantitative and no interferences
were introduced. The method was used for identification of selenoprotein P after enrichment of the protein from human plasma.
Received: 28 June 1999 / Revised: 14 September 1999 / Accepted: 16 September 1999 |
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