Application of pressure cycling technology to tissue sample preparation for 2‐DE

Ringham, Heather N.; Bell, Richard L.; Smejkal, Gary B.; Behnke, James; Witzmann, Frank A.

doi:10.1002/elps.200600434

Cited by 12 publications

(11 citation statements)

References 6 publications

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“…PCT was performed using the Barocycler NEP-3229 instrument, disposable polypropylene PULSE Tubes FT-500 and the services of Pressure Biosciences (West Bridgewater, MA) as a research gift. The process was performed following suitable modification of standard protocol [18,19,21] for 60 cycles. Each cycle consisted of 20 s at 35 000 psi, followed by an instantaneous drop to atmospheric pressure and holding recovery.…”

Section: Pctmentioning

confidence: 99%

Strategies to recover proteins from ocular tissues for proteomics

et al. 2008

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We present here the results of protein extraction from different ocular regions using different detergents. Extraction strategies used to determine optimal protein extraction included: pressure cycling and aqueous-organic phase extraction in combination with electrophoretic fractionation for anterior, posterior, and peripapillary sclera. Detergent extraction of proteins from freshly enucleated porcine eyes (n = 8) showed significant differences for different eye regions. Protein yield ranged from 2.3 to 50.7 mg protein/mg for different ocular tissues, with the lens yielding the most protein. ASB-14 and Triton X-100 provided the best protein yields (n = 10) for anterior and posterior sclera. The spectrophotometric measurements for ASB-14 were not consistent with SDS-PAGE densitometry. A combination of 0.5% Triton X-100, 0.5% Tween-20, and 0.1% Genapol C-100 was found optimal for extraction from sclera. Proteins from different regions of the eye are best extracted with different detergents. The pressure cycling technology provided superior extraction compared to the other methods. Additional aqueous-organic phase partitioning enables superior fractionation when compared to SDS-PAGE alone. Organic phase fractionation is compatible with MS and allowed identification of 34, 71, and 77 proteins respectively from anterior, posterior, and peripapillary sclera. The extraction strategy may affect the final outcome in protein profiling by MS or by other methods.

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Section: Pctmentioning

confidence: 99%

Strategies to recover proteins from ocular tissues for proteomics

et al. 2008

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“…Several techniques have been described to ‘crack’ worms, but each suffers from one or more limitations. Pressure Cycling Technology (PCT) uses expensive equipment to expose samples to multiple rounds of combined freezing, mechanical shearing and elevated hydrostatic pressure (40 cycles 35,000 psi) [6; 28]. Complete disruption of all worm stages, including dauers, can be accomplished using this procedure but it requires mixing of animals with a SiC abrasive [29].…”

Section: Discussionmentioning

confidence: 99%

Breaking Caenorhabditis elegans the easy way using the Balch homogenizer: An old tool for a new application

Bhaskaran

Butler

Becerra

et al. 2011

Analytical Biochemistry

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The nematode Caenorhabditis elegans is a model organism best known for its powerful genetics. There is an increasing need in the worm community to couple genetics with biochemistry. Isolation of functionally-active proteins, or nucleic acids without the use of strong, oxidizing denaturants, or of sub-cellular compartments from C. elegans has, however, been challenging because of the worms' thick surrounding cuticle. The Balch Homogenizer is a tool that has found much use in mammalian cell culture biology. The interchangeable, single ball bearing design of this instrument permits rapid permeabilization, or homogenization, of cells. Here we demonstrate the utility of the Balch Homogenizer for studies with C. elegans. We describe procedures for the efficient breakage and homogenization of every larval stage, including dauers, and show that the Balch Homogenizer can be used to extract functionally-active proteins. Enzymatic assays for catalase and dihydrolipoamide dehydrogenase show that sample preparation using the Balch Homogenizer equals or out-performs conventional methods employing boiling, sonication or Dounce homogenization. We also describe phenol-free techniques for isolation of genomic DNA and RNA. Finally, we use the tool to isolate coupled mitochondria and polysomes. The reuseable Balch Homogenizer represents a quick and convenient solution for undertaking biochemical studies on C. elegans.

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“…PCT provides a simple, fast, effective and reproducible process to release cellular contents from biological samples [26,27,28]. Previously, it has been shown that the use of PCT increased protein yields from E. coli , where PCT extracted 14.2% more total protein than using a standard bead mill [29,30]. Furthermore, 2-DE showed 801 protein spots in the PCT lysate, compared to 760 spots in the bead mill lysate [29].…”

Section: Introductionmentioning

confidence: 99%

“…Furthermore, 2-DE showed 801 protein spots in the PCT lysate, compared to 760 spots in the bead mill lysate [29]. In mammalian liver tissue, PCT isolated more protein, as well as unique proteins when compared to protein isolation using a Polytron or ground-glass (GG) homogenizers [30,31]. Szabo et al found that PCT-assisted glycan release resulted in the rapid release of asparagine-linked glycans from bovine ribonuclease B, human transferrin and polyclonal human immunoglobulin [32].…”

Section: Introductionmentioning

confidence: 99%

Comparative Proteomic Analysis of Cotton Fiber Development and Protein Extraction Method Comparison in Late Stage Fibers

et al. 2016

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The distinct stages of cotton fiber development and maturation serve as a single-celled model for studying the molecular mechanisms of plant cell elongation, cell wall development and cellulose biosynthesis. However, this model system of plant cell development is compromised for proteomic studies due to a lack of an efficient protein extraction method during the later stages of fiber development, because of a recalcitrant cell wall and the presence of abundant phenolic compounds. Here, we compared the quality and quantities of proteins extracted from 25 dpa (days post anthesis) fiber with multiple protein extraction methods and present a comprehensive quantitative proteomic study of fiber development from 10 dpa to 25 dpa. Comparative analysis using a label-free quantification method revealed 287 differentially-expressed proteins in the 10 dpa to 25 dpa fiber developmental period. Proteins involved in cell wall metabolism and regulation, cytoskeleton development and carbohydrate metabolism among other functional categories in four fiber developmental stages were identified. Our studies provide protocols for protein extraction from maturing fiber tissues for mass spectrometry analysis and expand knowledge of the proteomic profile of cotton fiber development.

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Application of pressure cycling technology to tissue sample preparation for 2‐DE

Cited by 12 publications

References 6 publications

Strategies to recover proteins from ocular tissues for proteomics

Strategies to recover proteins from ocular tissues for proteomics

Breaking Caenorhabditis elegans the easy way using the Balch homogenizer: An old tool for a new application

Comparative Proteomic Analysis of Cotton Fiber Development and Protein Extraction Method Comparison in Late Stage Fibers

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