Efficient isolation of intact lysosomes, peroxisomes and nuclei.

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. The 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

Kit Details and Example Data:

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 8,000 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 200 mg 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 tissue. Liver and kidney tissues (200 mg 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.
Peroxisome enrichment from tissue. Liver and kidney tissues (300 mg 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.
Nuclei enrichment from tissue. Liver and kidney tissue (400 mg 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.

Organelle enrichment from cells

Several cell lines were examined for lysosome isolation: A431 [American Type Culture Collection (ATCC* Resource Center), Product # CRL-1555], HeLa (ATCC, Product # CCL-2), and HepG2 cells (ATCC, Product # 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.

Cross-contamination of enriched organelles. Liver and kidney tissues (200 mg 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 10 µg 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), 233-244.
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), 1271-1280.
6. Lukong, K.E., et al. (2001). J. Biol Chem., 276, 46172-46181.
7. Van Veldhoven, P.P., et al. (1996). Anal Biochem, 237(1), 17-23.

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 Membrane Protein Extraction Reagent Kit

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