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Pressure Cycling Technology (PCT): a Novel Sample Preparation Approach to Biomarker Discovery and Drug Development in Lipid-Rich Samples
Analysis of protein expression from lipid-rich tissues is key to the development of a better understanding of obesity, an epidemic clinical condition directly related to roughly 300,000 deaths per year in the US, and associated with an increased risk for a number of serious diseases, including heart disease, stroke, cancer, and diabetes. Traditional detergent-based methods for protein extraction from lipid-rich samples can produce disappointing yields and fail to uncover the breadth of the adipocyte proteome. Moreover, these strong detergents often interfere with downstream analytical methods, such as gel electrophoresis, liquid chromatography, or mass spectrometry. ProteoSolveLRS, when used in combination with Pressure BioSciences’ pressure cycling technology (PCT), provides a powerful alternative to traditional detergent-based extraction methods. By combining the Power of PCT with the novel chemistry provided by the ProteoSolveLRS kit, it is possible to fractionate the lipid and protein components of a sample, resulting in higher protein recovery and greater reproducibility when compared to traditional, detergent-based extraction methods. In addition, proteins that are under-represented in extracts obtained by conventional methods have been successfully extracted by the PCT/ProteoSolveLRS method, as shown in the figure below. These novel proteins offer the potential to be important diagnostic or prognostic markers.
2D Gel Analysis of Proteins from Murine Adipose Tissue
 Protein Extracted by the PCT-Dependent Detergent-Free Method |
 Protein Extracted in 9M Urea, 4% CHAPS |
| Extraction of proteins from a 100 mg block of normal murine white adipose tissue. Extraction in the conventional CHAPS-based 2D sample extraction buffer (right) results in a solution of predominantly blood plasma proteins, while tissue dissolution by PCT and ProteoSolveLRS (left) followed by removal of lipids and solvent and reconstitution in 2D electrophoresis sample buffer appears to produce a sample representing the entire proteome of the adipose tissue. | |
The simultaneous method of tissue disruption, liquid-liquid partitioning, and protein extraction enabled by ProteoSolveLRS, in combination with the PCT Sample Preparation System (PCT SPS), is the first automated, reproducible, detergent-free method for protein extraction from lipid-rich samples. This novel method uses an instrument (Barocycler) to generate alternating cycles of hydrostatic pressure between ambient and 35,000 psi to modulate the mutual solubility of the organic solvents included in the ProteoSolveLRS kit. This process promotes improved solubilization and fractionation of the various sample constituents. Tissue samples and ProteoSolveLRS reagents are added to single-use processing vials called PULSE Tubes™ (Pressure Used to Lyse Samples for Extraction), which are then placed into the reaction chamber of the Barocycler instrument. Subsequently, these specially designed vials transmit hydrostatic pressure to the sample, while keeping the sample fully enclosed to eliminate cross-contamination and to minimize operator exposure to biohazardous 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 exposure to cycles of high hydrostatic pressure and rapid de-pressurization, cells are ruptured and dissolved in a solvent mixture, which is rendered nearly homogenous under cycled pressure. Subsequently, immiscible solvent phases separate, fractionating the sample constituents according to their inherent solubility in respective solvent phases.
Features & Benefits:
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Specifications of the ProteoSolveLRS Kit
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*For Research Use Only
Note: The kit does not include reagents for the solvent removal step.
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