Convenient and versatile subcellular extraction procedure, that facilitates classical protein expression profiling and functional protein analysis.
Standardized sample preparation to reduce proteome complexity facilitates subsequent proteome analysis. Here we describe a robust sequential extraction method that enables simple fractionation of proteins in their native state according to their subcellular localization, yielding four subproteomes enriched in (a) cytosolic; (b) membrane and membrane organelle-localized; (c) soluble and DNA-associated nuclear and (d) cytoskeletal proteins. Efficiency and selectivity is demonstrated by morphological-, two-dimensional electrophoresis image-, immunological- as well as enzymatic-analysis. In pilot studies, subcellular redistribution of regulatory proteins was successfully measured.
Abdolzade-Bavil
Afsaneh
A
Merck, Life Science Products R&D, Darmstadt, Germany.
Hayes
Scott
S
Goretzki
Lothar
L
Kröger
Michaela
M
Anders
Jonas
J
Hendriks
Robertus
R
D015153:Blotting, Western; D002458:Cell Fractionation / Q000379:methods* / Q000592:standards*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000737:chemistry; D002477:Cells / Q000187:drug effects; D003598:Cytoskeletal Proteins / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004798:Enzymes / Q000032:analysis / Q000378:metabolism; D006801:Humans; D007525:Isoelectric Focusing; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000494:pharmacology; D015388:Organelles / Q000737:chemistry; D010865:Pilot Projects; D011506:Proteins / Q000378:metabolism*; D020543:Proteome / Q000032:analysis*; D012680:Sensitivity and Specificity; D013347:Subcellular Fractions / Q000737:chemistry*
D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D004798:Enzymes; D008565:Membrane Proteins; D016328:NF-kappa B; D011506:Proteins; D020543:Proteome