Modification of the immobilized metal affinity electrophoresis using sodium dodecyl sulfate polyacrylamide gel electrophoresis.
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 pI 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.
Lee
Bao-Shiang
BS
Protein Research Laboratory, Research Resources Center, University of Illinois, Chicago, IL, USA. boblee@uic.edu
Jayathilaka
G D Lasanthi P
GD
Huang
Jin-Sheng
JS
Decresce
David
D
Borgia
Jeffrey A
JA
Zhou
Xiaofeng
X
Gupta
Shalini
S
D000535:Aluminum; D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation*; D006801:Humans; D007501:Iron; D008345:Manganese; D008670:Metals; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000032:analysis; D010748:Phosphopeptides / Q000032:analysis / Q000187:drug effects; D010750:Phosphoproteins / Q000032:analysis* / Q000187:drug effects; D012709:Serum Albumin / Q000032:analysis; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*; D014025:Titanium
D002364:Caseins; D008670:Metals; D010748:Phosphopeptides; D010750:Phosphoproteins; D012709:Serum Albumin; D012967:Sodium Dodecyl Sulfate; D008345:Manganese; D010047:Ovalbumin; D000535:Aluminum; D014025:Titanium; D007501:Iron