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Organelle Enrichment Kits


Efficient isolation of intact lysosomes, peroxisomes and nuclei.

Organelle Enrichment Kits

Thermo Scientific Organelle Enrichment Kits are for subcellular fractionation and isolation of intact lysosomes, peroxisomes and nuclei by high-speed density gradient centrifugation for proteomics analysis.

These kits enable 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.

Highlights:

  • 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

Product Details:

Thermo Scientific Organelle Enrichment Kits.
Kit Sample Source OptiPrep™
Density Gradient
Centrifugation
Speed (x g)
Centrifugation
Time (Minutes)
Lysosome  Cells 15%, 17%, 20%,
23%, 27% and 30%
145,000 120
Tissue (soft & hard)
Peroxisome Soft Tissue 27.5%, 30% and 35% 180,000 90
Hard Tissue 18%, 20% and 27.5% 180,000 90
Nuclei Tissue (soft & hard) 7%, 23% and 27.5% 40,000 90

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.

Lysosome enrichment from cultured cells. Approximately 200mg of wet cell paste was processed from A431 and HeLa cells using the Lysosome Enrichment Kit for Tissue and Cultured Cells. Total cell lysate and isolated lysosomes were analyzed by Western blotting for Lamp-1 and Cathepsin D, membrane-bound and soluble lysosome markers, respectively. Lysosome enrichment from cultured cells.

Lysosome enrichment from tissue. Liver and kidney tissues (200mg each) were processed using the Lysosome Enrichment Kit for Tissue and Cultured Cells. Total lysate and isolated lysosomes were analyzed by Western blotting for Lamp-1, a lysosomal membrane protein marker. Lysosome enrichment from tissue.
Peroxisome enrichment from tissue. Liver and kidney tissues (300mg each) were processed using the Peroxisome Enrichment Kit for Tissue. Total lysate and isolated peroxisomes were analyzed by Western blotting for PMP70, a peroxisomal membrane protein marker. Peroxisome enrichment from tissue.
Nuclei enrichment from tissue. Liver and kidney tissue (400mg each) were processed using the Nuclei Enrichment Kit for Tissue. Total cell lysate and isolated nuclei were analyzed by Western blotting for histone deacetylase (HDAC), a soluble nuclear marker. Nuclei enrichment from tissue.

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 50-200mg 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.

Cross-contamination of enriched organelles.
Cross-contamination of enriched organelles. Liver and kidney tissues (200mg each) were processed using each Thermo Scientific Organelle Isolation Kit and analyzed via Western blotting for contamination by probing for Cox4 and PMP70, mitochondria and peroxisome markers, respectively. Samples were normalized by protein amount (approximately 10µg of total proteins), except for the liver and kidney fractions from the peroxisome experiment, which were normalized by volume. Tissues from rats of identical weight and ages were also processed with similar kits from Competitor S according to the manufacturer's instructions. Significant contamination in the lysosome and nuclei fractions is observed with the procedure from Competitor S.

References:

  1. Gjoen, T., et al. (1997). Lysosomes and endocytosis. Subcellular fractionation: A practical approach. Eds. Graham, J.M. and Rickwood, D., IRL Press Limited, Oxford, England, 169-200.
  2. Graham, J.M. (1997). Homogenization of tissues and cells. Subcellular fractionation: A practical approach. Eds. Graham, J.M. and Rickwood, D., IRL Press Limited, Oxford, England, 1-28.
  3. Hajra, A.K., et al. (1985). Anal Biochem. 148(2):233-44.
  4. Hinton, R.H. and Mullock, B.M. (1997). Isolation of subcellular fractions. Subcellular fractionation: A practical approach. Eds. Graham, J.M. and Rickwood, D., IRL Press Limited, Oxford, England, 31-69.
  5. Luers, G., et al. (1993). J. Cell. Bio. 121(6):1271-80.
  6. Lukong, K.E., et al. (2001). J. Biol Chem. 276:46172-81.
  7. Van Veldhoven, P.P., et al. (1996). Anal Biochem 237(1):17-23.

Notice:

The following products are scheduled to be discontinued January 2015:

Peroxisome Enrichment Kit for Tissue
(Part No. 89840)
and
Nuclei Enrichment Kit for Tissue
(Part No. 89841)

Related Resources:

Review of cell fractionation and organelle isolation

Related Products:

Mitochondrial Isolation Kit for Cultured Cells
Mitochondrial Isolation Kit for Tissue
Subcellular Protein Fractionation Kit
NE-PER Nuclear and Cytoplasmic Extraction Reagents
Mem-PER Plus Membrane Protein Extraction Kit


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
89839 Lysosome Enrichment Kit for Tissues and Cultured Cells
Formulation: Multi-component kit
Sufficient For: 25 samples each containing 50 to 200mg of cells or tissue

Kit Contents:
Lysosome Enrichment Reagent A, 90mL
Lysosome Enrichment Reagent B, 90mL
OptiPrep Cell Separation Media, 50mL
PBS Pack (makes 500mL), 1 pack
230mL kit Product Instructions for product #89839 Lysosome Enrichment Kit for Tissues and Cultured Cells MSDS for product #89839 Lysosome Enrichment Kit for Tissues and Cultured Cells Certificate of Analysis for product #89839 Lysosome Enrichment Kit for Tissues and Cultured Cells $298.00

Add to cart
89840 Peroxisome Enrichment Kit for Tissue
Formulation: Multi-component kit
Sufficient For: 25 samples each containing 50 to 300mg of soft or hard tissue

Kit Contents:
Peroxisome Enrichment Reagent A, 90mL
Peroxisome Enrichment Reagent B, 90mL
OptiPrep Cell Separation Media, 50mL
PBS Pack (makes 500mL), 1 pack
230mL kit Product Instructions for product #89840 Peroxisome Enrichment Kit for Tissue MSDS for product #89840 Peroxisome Enrichment Kit for Tissue Certificate of Analysis for product #89840 Peroxisome Enrichment Kit for Tissue $291.00

Add to cart
89841 Nuclei Enrichment Kit for Tissue
Formulation: Multi-component kit
Sufficient For: 25 samples each containing 400 to 500mg of soft or hard tissue

Kit Contents:
Nuclei Enrichment Reagent A, 90mL
Nuclei Enrichment Reagent B, 90mL
OptiPrep Cell Separation Media, 50mL
PBS Pack (makes 500mL), 1 pack
230mL kit Product Instructions for product #89841 Nuclei Enrichment Kit for Tissue MSDS for product #89841 Nuclei Enrichment Kit for Tissue Certificate of Analysis for product #89841 Nuclei Enrichment Kit for Tissue $310.00

Add to cart
89917 Lamp-1 Monoclonal Antibody
Organelle target: Lysosome
50µg MSDS for product #89917 Lamp-1 Monoclonal Antibody $217.00

Add to cart

Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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