Thermo Fisher Scientific Lysosome Enrichment Kit for Tissues and Cultured Cells

Thermo Fisher Scientific Lysosome Enrichment Kit for Tissues and Cultured Cells

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

Features

• Efficient and easy to use — gradient centrifugation separates organelles from contaminants
• Compatible — suitable for various downstream applications (2D/MS, EM, profiling, expression, signaling, localization)
• Complete — sufficient reagents for 25 preparations

Organelle Enrichment from Tissue

Six- to eight-week-old female Sprague-Dawley rats (average weight 160 g) were fasted overnight before sacrifice. Liver and kidneys were excised, washed with ice-cold PBS, minced, and homogenized in ice-cold Reagent A using a Polytron™ Tissue Tearer for 45 seconds at 8000 rpm. Reagent B was added and the homogenate centrifuged to remove debris. The supernatant was layered on a discontinuous OptiPrep™ gradient and centrifuged to isolate the targeted organelle. The target band was collected and analyzed by Western blotting.

Organelle Enrichment from Cells

Cell lines used:

• A431 (ATCC #CRL-1555)
• HeLa (ATCC #CCL-2)
• HepG2 (ATCC #HB-8065)

Cells were grown to 80–90% confluency, harvested (50–200 mg wet cell paste), and processed for lysosome enrichment. Ice-cold Lysosome Reagent A was added and lysis performed using a Misonix Sonicator 3000 (15 pulses, 15W). Lysosome Reagent B was added, and the homogenate centrifuged to remove debris. The supernatant was layered on discontinuous OptiPrep gradients and centrifuged to isolate the target organelle. The target band was collected and analyzed by Western blotting, normalized by protein amount.

Related Products

• Mitochondria Isolation Kit for Cultured Cells
• Mitochondria Isolation Kit for Tissue

Specifications

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