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Modification of the immobilized metal affinity electrophoresis using sodium dodecyl sulfate polyacrylamide gel electrophoresis
Author(s) -
Lee BaoShiang,
Jayathilaka G. D. Lasanthi P.,
Huang JinSheng,
Decresce David,
Borgia Jeffrey A.,
Zhou Xiaofeng,
Gupta Shalini
Publication year - 2008
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200800024
Subject(s) - chemistry , electrophoresis , gel electrophoresis , chromatography , polyacrylamide gel electrophoresis , gel electrophoresis of proteins , sodium dodecyl sulfate , phosphate , metal , gel electrophoresis of nucleic acids , phosphate buffered saline , affinity electrophoresis , metal ions in aqueous solution , ovalbumin , extraction (chemistry) , biochemistry , affinity chromatography , biology , enzyme , organic chemistry , immune system , immunology
Modification to the original immobilized metal affinity electrophoresis (IMAEP) technique is presented. SDS‐PAGE is used instead of native PAGE for improved extraction of phosphoproteins from a mixture of proteins. Protein samples treated with 2% w/v SDS instead of native sample buffer ensure that proteins are negatively charged. These negative charges on the proteins assure that the proteins migrate electrophoretically towards the anode regardless of their p I values and hence pass through the region embedded with the metal ions. Another benefit of treating proteins with SDS is that it unfolds the phosphoproteins exposing the phosphate groups to facilitate the metal‐phosphate interactions. Phosphorylated ovalbumin can only be extracted after SDS sample buffer treatment. Data show that there is no detrimental effect upon SDS treatment on the extraction of phosphoproteins from a mixture of proteins. Electrophoretic migration of phosphoproteins ceases upon encounter with metal ions like Al +3 , Ti +3 , Fe +3 , Fe +2 , and Mn +2 whereas non‐phosphorylated proteins migrate freely.