One major challenge in functional proteomics is the separation of complex protein mixtures to allow detection of low abundance proteins and provide for quantitative and qualitative analysis of proteins impacted by environmental parameters. Prerequisites for the success of such analysis are standardized and reproducible procedures for sample preparation prior to 1 or 2 DGE and/or liquid chromatography (LC). Due to the complexity of total proteomes and the divergence of protein properties it is required to prepare standardized partial proteomes of a given organism in order to maximize the coverage of the proteome and to increase the chance to visualize low-abundance proteins. The unique ProteoExtract® Subcellular Proteome Extraction Kit (S-PEK) enables the differential extraction of proteins according to their subcellular localization. The special mild S-PEK procedure yields proteins in their native state making it highly suited for demanding proteomics applications including enzyme activity assays e.g. reporter gene assays and subcellular redistribution assays to monitor protein shuttling e.g. singnaling proteins. Optimized protocols are available for freshly prepared tissue culture cells, frozen cell pellets and fragmented tissues. The kit contains four Extraction Buffers prepared with ultra-pure chemicals to ensure high reproducibility, protease inhibitor cocktail to prevent protein degradation and Benzonase® nuclease o remove contaminating nucleic acids. tS-PEK takes advantage of the differential solubility of certain subcellular compartments in special reagent mixtures preserving the structural integrity of the subcellular structures before and during the extraction. For suspension-grown cells the S-PEK extraction starts with gentle sedimentation and washing of the cells. For frozen cells and fragmented tissues, extraction starts with resuspension in the first extraction buffer. In case of adherent cells, the S-PEK procedure is performed directly in the tissue culture dish. The cells or the parts of the cells remain attached to the plate during the entire extraction procedure until the appropriate extraction reagent is used. Thus, the early destruction of the cellular structure by enzymatic or mechanical detachment of cells from the tissue culture plate and any mixing of different subcellular compartments is prevented. A schematic overview of the S-PEK extraction procedure applied to adherent tissue culture cells is shown in Fig 1 A and morphological changes of the cells are documented. ProteoExtract®Subcellular Proteome Extraction Kit yields the total proteome fractionated into four sub proteomes of decreased complexity. With Extraction Buffer I cytosolic proteins are released (fraction 1). Subsequently, membranes and membrane organelles are solubilized with Extraction Buffer II, without impairing the integrity of nucleus and cytoskeleton (fraction 2). Next, nucleic proteins are enriched with Extraction Buffer III (fraction 3). Components of the cytoskeleton are finally solubilized with Extraction Buffer IV (fraction 4).