A subcellular prefractionation protocol for minute amounts of mammalian cell cultures and tissue.
Subcellular localization represents an essential, albeit often neglected, aspect of proteome analysis. Generally, the subcellular location of proteins determines the function of cells and tissues. Here we present a robust and versatile prefractionation protocol for mammalian cells and tissues which is appropriate for minute sample amounts. The protocol yields three fractions: a nuclear, a cytoplasmic, and a combined membrane and organelle fraction. The subcellular specificity and the composition of the fractions were demonstrated by immunoblot analysis of five marker proteins and analysis of 43 proteins by two-dimensional gel electrophoresis and mass spectrometry. To cover all protein species, both conventional two-dimensional and benzyldimethyl-n-hexadecyl ammonium chloride-sodium dodecyl sulfate (16-BAC-SDS) gel electrophoresis were performed. Integral membrane proteins and strongly basic nuclear histones were detected only in the 16-BAC-SDS gel electrophoresis system, confirming its usefulness for proteome analysis. All but one protein complied to the respective subcellular composition of the analyzed fractions. Taken together, the data make our subcellular prefractionation protocol an attractive alternative to other prefractionation methods which are based on less physiological protein properties.
Guillemin
Isabelle
I
Abteilung Tierphysiologie, Technische Universität Kaiserslautern, 67653 Kaiserslautern, Germany.
Becker
Michael
M
Ociepka
Kornelia
K
Friauf
Eckhard
E
Nothwang
Hans Gerd
HG
D000818:Animals; D001923:Brain Chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D015151:Immunoblotting; D008297:Male; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis; D009687:Nuclear Proteins / Q000032:analysis; D015388:Organelles / Q000737:chemistry; D020543:Proteome / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000737:chemistry
D008565:Membrane Proteins; D009687:Nuclear Proteins; D020543:Proteome