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Syn-PER™ Synaptic Protein Extraction Reagent (Thermo Scientific™)

Thermo Scientific Syn-PER Synaptic Protein Extraction Reagent is a proprietary cell lysis reagent for efficient isolation of synaptosomes containing functional synaptic proteins from neuronal tissue and primary cultured neurons.

Features of Syn-PER Synaptic Protein Extraction Reagent:

Efficient synapse extraction—obtain up to 10 µg of synaptic protein per milligram of neuronal tissue or 4 µg synaptic protein per 35mm dish of primary cultured neurons (106 cells)
Gentle formulation—isolate viable synaptosomes; then lyse these synapses to extract native synaptic proteins and preserve phosphoprotein integrity
Fast procedure—obtain synaptosomal suspension of intact synapses in less than one hour
Simple protocol—requires no ultracentrifugation steps

Applications of Syn-PER Synaptic Protein Extraction Reagent:
• Isolate functional synaptosomes to study neurotransmitter release
• Extract pre- and post-synaptic proteins to identify changes in protein composition and function in synapses
• Preserve and study labile or transient protein phosphorylation events associated with synapses

Syn-PER Reagent is used to prepare synaptosomes containing biologically active pre-and post-synaptic proteins (i.e., intact membranes and protein complexes of synapses). When used with fresh neuronal tissue or primary cultured neurons, synaptosomes prepared with the Syn-PER Reagent can be used to study synaptic transmission. The synaptosomal proteins contained in these extracts can also be used for downstream applications such as Western blotting, immunoprecipitation, enzymatic activity assays and protein-protein interaction studies. Syn-PER Synaptic Protein Extraction Reagent minimizes the degradation of phosphoproteins and is ideal for studies requiring the preservation of phosphoprotein integrity.

Syn-PER Synaptic Protein Extraction Reagent efficiently enriches pre-and post-synaptic protein within synaptosomes with high yield. The nondenaturing cell lysis reagent is compatible with many downstream applications, including neurotransmitter release assays, enzyme assays (e.g., phosphatase, kinases), immunoassays, various chromatography procedures, and electrophoresis. In addition, Syn-PER Reagent preserves phosphoprotein integrity better than most commercially available extraction buffers, even in the absence of phosphatase inhibitors. Syn-PER Reagent does not contain protease or phosphatase inhibitors. However, inhibitors such as Thermo Scientific Halt Protease and/or Phosphatase Inhibitors can be added just before use to prevent proteolysis or to offer additional protection from the high phosphatase activity normally present in brain tissue.

Synaptosomes are isolated nerve terminals that are generated during the homogenization of nerve tissue. Synaptosome preparations can be used for extraction of pre- and post-synaptic proteins because they contain the complete pre-synaptic terminal, including mitochondria and synaptic vesicles, along with the post-synaptic membrane and the post-synaptic density. Synaptosomes are commonly used to study synaptic function because they contain functional ion channels, receptors, enzymes and intact synaptic vesicles, which can take up and release neurotransmitters. Synaptosome preparation typically involves several ultracentrifugation steps and density gradients to separate this cell fraction from other organelles.

More Product Data
Method to isolate functional synaptosomes

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Mitochondria Isolation Kit for Tissue (Thermo Scientific™)

The Thermo Scientific Mitochondria Isolation Kit for Tissue enables isolation of intact mitochondria from soft and hard tissue samples in about an hour using reagent-based or Dounce homogenization methods.

Features of the Mitochondria Isolation Kit for Tissue:

Quick and Convenient—isolate intact mitochondria in less than 60 minutes
Versatile—offers two methods of isolation from both soft and hard tissue samples
Multi-sample format—reagent-based approach enables simultaneous processing of multiple samples
Optional alternate method—reagents and protocol included for the traditional Dounce homogenization procedure with fewer required strokes
Bench-top compatible—both procedures can be performed using a microcentrifuge tube

This kit provides for two methods for mitochondria isolation. The first method uses a unique reagent-based procedure that enables simultaneous multi-sample processing. The second method relies on traditional Dounce homogenization for tissue disruption and subsequent isolation of the organelle. Both procedures use differential centrifugation to separate the intact mitochondria using a bench-top microcentrifuge and are completed in less than 60 minutes. In addition, both procedures have been optimized for maximum yield of mitochondria with minimal damage to integrity. The isolated mitochondria may be used for a number of downstream applications, including 1D and 2D Western blotting and protein profiling using mass spectroscopy.

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Lysosome Enrichment Kit for Tissues and Cultured Cells

Mitochondria Isolation Kit for Cultured Cells (Thermo Scientific™)

The Thermo Scientific Mitochondria Isolation Kit for Cultured Cells provides a versatile, microcentrifuge-tube method for fractionating intact mitochondria from cultured mammalian cell samples in about 40 minutes.

Features of the Mitochondria Isolation Kit for Cultured Cells:

Fast—isolate intact mitochondria from 20 million cells in approximately 40 minutes (post-cell harvest)
Flexible—process several samples simultaneously using the reagent-based method, or obtain highest possible yield with the Dounce homogenization method
Optimizable—guidelines provided for optimizing purity vs. yield with the reagent-based method
Benchtop-easy—isolation performed in a microcentrifuge tube
Compatible—isolated mitochondria may be lysed with detergent or processed for almost any downstream application, including 2D electrophoresis

Isolation of mitochondria is typically a laborious process requiring single-sample processing with Dounce homogenization. The Mitochondria Isolation Kit uses a non-mechanical, reagent-based method that allows multiple (six) cultured cell samples to be processed concurrently in about 40 minutes. Cultured mammalian cell pellets are gently lysed using a proprietary formulation that results in maximum yield of mitochondria with minimal damage to integrity.

The product instructions describe guidelines for optimizing purity vs. yield parameters. The kit also offers an optimized Dounce homogenization procedure, which results in two-fold greater mitochondria recovery compared to the reagent-based method. Both methods use differential centrifugation to separate the mitochondrial and cytosolic fractions with a benchtop microcentrifuge and are completed in approximately 40 minutes (post-cell harvest). Once isolated, the mitochondria can be used in downstream applications such as apoptosis, signal transduction and metabolic studies, as well as to facilitate mitochondrial proteomics efforts.

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Lysosome Enrichment Kit for Tissues and Cultured Cells

Lysosome Enrichment Kit for Tissues and Cultured Cells (Thermo Scientific™)

The Thermo Scientific™ Lysosome Enrichment Kit for Tissue and Cultured Cells enables isolation and enrichment for intact lysosomes from crude cell and tissue extracts. The kit provides sufficient reagents for preparing 25 extracts and uses OptiPrep Cell Separation Media for the density-based separation of lysosomes from contaminating cell structures. The isolated lysosomes may be used for a number of downstream applications, including 2D/MS for proteomics research, electron microscopy, disease profiling and gene expression, signal transduction, and interaction or localization.

Features of the Lysosome Enrichment Kit for Tissue and Cultured Cells:

Efficient and easy to use—kit reagents and gradient centrifugation separate organelles from contaminating structures
Compatible—prepare samples for downstream applications, including 2D/MS, electron microscopy, disease profiling, gene expression, signal transduction and interaction or localization studies
Complete—kits contain sufficient material for 25 applications

This kit enables enrichment of intact organelles from cells and tissue. Each kit uses density gradient centrifugation to separate organelles from contaminating cellular structures. The isolated organelles may be used for a number of downstream applications, including 2D/MS, electron microscopy, disease profiling, gene expression, signal transduction and interaction or localization studies.

Organelle enrichment from tissue:
Six- to eight-week-old female Sprague-Dawley rats with an average weight of 160 g were fasted overnight before sacrifice. The liver and kidneys were excised, washed with ice-cold PBS, minced using surgical scissors and then homogenized in ice-cold Reagent A using a Polytron™ Tissue Tearer for 45 seconds at 8000 rpm. Reagent B was subsequently added and the homogenate was centrifuged to remove cellular debris. The resulting supernatant was overlayed on a discontinuous gradient of OptiPrep™ Cell Separation Media and centrifuged to isolate and enrich the targeted organelle. The target band was removed from the gradient and analyzed by Western blotting.

Organelle enrichment from cells:
Several cell lines were examined for lysosome isolation: A431 [American Type Culture Collection (ATCC™ Resource Center), #CRL-1555], HeLa (ATCC, #CCL-2), and HepG2 cells (ATCC, #HB-8065). The cells were grown to 80-90% confluency. Approximately 50-200 mg of wet cell paste was harvested and processed for lysosome enrichment. Ice-cold Lysosome Reagent A was added to the cells and lysis was performed using a Misonix Sonicator 3000 with 15 pulses delivering 15W of power.

Subsequently, Lysosome Reagent B was added, and the homogenate was centrifuged to remove cellular debris. The resulting supernatant was overlayed on several discontinuous gradients of the OptiPrep Cell Separation Media and centrifuged to isolate and enrich for the target organelle. The target band was removed from the gradient and, along with total lysate, normalized by protein amount (unless otherwise noted) and analyzed by Western blotting.

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