Direct determination of selenoproteins in polyvinylidene difluoride membranes by electrothermal atomic absorption spectrometry.
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.
Department of Analytical and Pharmaceutical Chemistry, The Royal Danish School of Pharmacy, Copenhagen. ulsi@mail.dfh.dk
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U
Sidenius
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Gammelgaard
D002138:Calibration; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D016014:Linear Models; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins / Q000032:analysis*; D051149:Selenoprotein P; D051140:Selenoproteins; D012680:Sensitivity and Specificity; D013054:Spectrophotometry, Atomic / Q000379:methods*; D013696:Temperature; D005465:Fluorocarbon Polymers
D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins; D051149:Selenoprotein P; D051140:Selenoproteins; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers