ProteoExtract® Partial Proteome Extraction Kits

Novagen

Proteome analysis implies the ability to separate proteins with high resolution and reproducibility prior to characterization by mass spectrometry, microsequencing or equivalent means. Currently, two-dimensional-electrophoresis (2DE) remains the highest resolution technique for protein separation when complex samples need to be arrayed for analysis. The sample preparation of any protein mixture for subsequent 2DE is of major importance, as it will affect the overall performance of the technique. In general, pretreatment of samples involves solubilization, denaturation and reduction of proteins in the presence of special reagents. The prerequisites for an efficient sample preparation method are reproducible solubilization of all types of proteins, prevention of protein degradation as well as a thorough removal of contaminating nucleicacids e.g. by enzymatic digestion. Due to their very specific characteristics, some proteins may be welldenatured and solubilized by a given detergent or chaotrope, whereas other proteins will require another reagent. Consequently, the future of solubilization is to find mixtures of detergents and chaotropes able to cope with the diversity of proteins encountered in the complex samples to be separated by 2DE. Additionally each biological sample requires special procedures e.g. for cell disruption. For this reason, we have developed the Mammalian Partial Proteome Extraction Kit (P-PEK) as a tool to extract proteins of variable solubility from both tissue culture cells and dissected mammalian tissues in the presence of different detergents and chaotropes. P-PEK is designed for serial sample preparation of complex protein mixtures using reagent mixtures with increasing solubilization strength. Each of the provided Extraction Reagents solubilizes a different subset of cellular proteins. The most soluble proteins of mammalian cells are released by mechanical disruption of the cells in Extraction Reagent 1 (Fraction 1). Proteins of intermediate solubility are subsequently extracted with Extraction Reagent 2 (Fraction 2). For solubilization of proteins otherwise insoluble in Extraction Reagent 2 the kit provides Extraction Reagent 3 with a special formulation for efficient membrane protein extraction (Fraction 3). The resulting fractions of proteins subsequently extracted with Extraction Reagent 1, 2 and 3 can be directly analyzed on separate 2DE-gels leading to an increased number of spots to be visualized in total. Finally the proteins still insoluble in Extraction Reagents 3 can be solubilized using the provided SDS-Buffer that can beanalyzed by one-dimensional SDS-PAGE (Fraction 4). A schematic representation of P-PEK as wells corresponding 2D-gels using human Hep G2 tissue culture cells are shown in Figure 1. aTo get optimal resolution and reproducibility in 2DE-protein patterns, the kit contains reagents composed of ultrapure chemicals. In order to preserve the protein profile, a ready-to-use Protease Inhibitor Cocktail is added. To reduce sample viscosity and increase spot resolution, Benzonase, a proprietary non-specific nuclease, is included for efficient nucleic acid degradation. Reduction of extracted proteins is performed with DTT. Despite that DTT is negatively charged at alkaline pH, itwas observed to be superior over TBP in the P-PEK procedure. Furthermore, besides being more difficult to handle due to spontaneous decomposition, TBP was found to be unstable in concentratedurea solutions as used in sample preparation for 2DE [1]

Prior to sequential extraction, lysis conditions are very important for the success of the sample preparation and they depend strongly on the cell type used. To allow for an efficient release of easily soluble proteins, tissue culture cells as well as mammalian tissues are sufficiently disrupted by homogenization in Extraction Reagent 1 using a combination of a glass homogenizer and theapplication of shearing force by passing the cell suspension through a narrow needle. Special extraction procedures have been developed and are described below. Independent of the sample preparation method chosen, it is most important to minimize protein modifications that might result in artefactual spots on 2DE-maps. Reagents and samples contain urea. For this reason, heating over +30 °C must be avoided as this may introduce considerable charge heterogeneity due to carbamoylation of the proteins by isocyanate formed from the decomposition of urea. Due to the importance of temperature control during sample preparation, the P-PEK-protocols formammalian samples work without any sonication step to avoid impairment of the poor temperature control when sonicating small volumes. Use the information given in table 1 as a guideline to score for the success of your extraction procedure.

Download Novagen01 – Calbiochem_PartialMammProtExtrKit

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