A Pressure Enhanced Systems Biology Kit
Simultaneous Extraction of Proteins, Lipids and Nucleic Acids from Cells and Tissues

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To fully characterize and understand disease states and to develop and evaluate drug therapies, it is essential to quantify and analyze proteins, lipids, nucleic acids and their interactions. The study of these complex interactions is referred to as Systems Biology, which incorporates proteomics, lipidomics, genomics and transcriptomics. Samples used in these studies are often limited, rare and extremely valuable. Therefore, analytes, such as proteins, lipids and nucleic acids, must be efficiently extracted from such samples so that as much information as possible is obtained. However, most sample preparation and extraction methods are not optimized for efficient recovery of total protein, isolation of lipids, and the simultaneous extraction of intact DNA and RNA. In addition, traditional methods may not adequately dissociate hydrophobic proteins that are tightly associated with membrane lipids. As a result, these proteins are often discarded in the insoluble fraction after tissue extraction, resulting in a bias toward the more readily soluble proteins. Traditional detergent-based protein extraction methods are not readily compatible with recovery of intact RNA and genomic DNA, and methods optimized for nucleic acid extraction are often incompatible with efficient protein recovery. Unfortunately it is not always possible to obtain multiple samples for multiple analyte extractions, and, dividing samples into several aliquots may significantly reduce sensitivity and, thus, the effectiveness of the analyses. This is particularly true for unique human samples, which are may be irreplaceable. To meet the growing need for a method to extract multiple, diverse analytes from the same sample, Pressure BioSciences has developed ProteoSolve-SB, a pressure enhanced Systems Biology kit for the simultaneous extraction of proteins, lipids, DNA and RNA from cells or tissues.

Proteins Extracted from Various Flash Frozen Tissues. Evaporation method (E), Precipitation method with Reagent C (P)	Phospholipid and Triglyceride Profiles of Lipid Phase by MALDI-TOF
DNA (Left) and RNA (Right) Extracted from Various Flash Frozen Tissues. Brain (Br), Cardiac Muscle, (Ca), Abdominal Fat Pad (Ad), Liver (Li) and Kidney (Kd)

The ProteoSolve-SB kit is designed for concurrent, detergent-free extraction and fractionation of proteins, lipids and nucleic acids from cells and tissues. Following extraction, the sample is separated by centrifugation into three fractions - a lipid-containing upper phase, a protein-containing lower phase, and an insoluble fraction (pellet and interface), which contains the DNA and RNA - as well as a small amount of protein. The dissolved sample proteins can be isolated from the lower phase by either evaporating the volatile solvent, or by protein precipitation with Reagent C. The resulting protein pellet can then be reconstituted in Reagent D for 2D electrophoresis or in another suitable reagent for downstream protein analysis by SDS-PAGE or other applications. Due to the strong chaotropic properties of Reagent A, most proteins are irreversibly denatured and do not retain any enzymatic activity and may no longer be recognized by antibodies generated against native protein structure. The lipids, in the upper phase, can be subjected to further separation or can be analyzed directly. The DNA and/or RNA can be isolated from the residual solid fraction by a number of standard methods or kits. Cells or tissue samples and ProteoSolve-SB reagents are added to single-use processing vials, called PULSE TubesT (Pressure Used to Lyse Samples for Extraction), which are then placed into the reaction chamber of the Barocycler, the high pressure instrument. These specially designed vials transmit hydrostatic pressure to the sample, while keeping the sample fully enclosed to eliminate cross-contamination and minimize operator exposure to hazardous materials. PBI currently offers two models of Barocyclers: the NEP3229, which accepts up to three PULSE Tubes at a time, and the smaller NEP2320, which processes one PULSE Tube per run. During cycles of high hydrostatic pressure and rapid de-pressurization, cells are ruptured and dissolved in the solvent mixture, which becomes nearly homogenous under pressure. Upon de-pressurization, solvent phases separate, fractionating the sample constituents according to their inherent solubility.

Features & Benefits:

Detergent-Free Extraction Automated Bench-Top Instrument Process Cells & Tissues Process Organelles & Membranes Improve Reproducibility

Increase Protein Recovery Identify Novel Proteins Direct Lipid Profiling Isolate DNA and RNA Discover Biomarkers

Specifications of the ProteoSolve_SB Kit

Kit Components PULSE Tubes Set of 12 2mL Sample CollectionTubes (RNase/DNase-free) 36 Reagent A - Extraction Solvent 1 Vial 20 mL Reagent B - Partitioning Reagent 1 Vial 20 mL Reagent C - Precipitation Reagent (optional) 1 Bottle 60 mL Reagent D - ProteoSolve IEF Reagent (optional) 1 Vial makes 25 mL (when reconstituted) Reagent E -  Ion Exchange Resin for Reagent D (optional) 1 Vial Reagent F - Reducing Agent for Reagent D (optional) 1 Vial Quick Guide 1 Each Instruction Manual 1 Each

Tissue Sample Amount/ PULSE Tube 10 - 200 mg Duration of Pressure Cycling Procedure 10 - 20 Minutes Compatible Methods of  Downstream Analysis HPLC, LC/MS, MALDI-TOF, GC/MS, SDS-PAGE, 2D electrophoresis, Western Blotting Number of Extractions/Kit 12 Storage Conditions 4-8oC, 1 year Laboratory Equipment Required Barocycler NEP 3229 or 2320 Microcentrifuge, SpeedVac Evaporator (optional)

*For Research Use Only

Note: The kit does not include reagents for the solvent removal step.

Relevant Literature:

1. Gross, V., et al., (2008). Tissue Fractionation by Hydrostatic Pressure Cycling: The Unified Sample Preparation Technique for Systems Biology Studies. Poster. ABRF 2008.
2. Schumacher, R.T., et al., (2002). An Automated Sample Preparation Solution for Nucleic Acid and Protein Extraction from Cells and Tissues. Am. Laboratory, 34, 38-43
3. Smejkal, G.B., et al., Sample preparation for two-dimensional gel electrophoresis using pressure cycling technology. Analytical Biochemistry. 2007, 363(2), pp. 309-311
4. Feng Tao, et al. (2006) Applications of Pressure Cycling Technology (PCT) in Proteomics in Separation Methods in Proteomics, Smejkal, G.B. and Lazarev, A., Eds., CRC Press, Taylor & Francis, Baton Rouge, pp. 3 -18.