Supernatants of cells were concentrated using a Microcon-10kDa Centrifugal Filter Unit with Ultracel-10 membrane (MRCPRT010, Millipore). The lysosome isolation kit (ab234047, Abcam) was used to isolate lysosomal fractions from cultured cells by differential centrifugation followed by density gradient centrifugation. Finally, the purified lysosomal fraction was obtained using an ultracentrifuge (145,000 × g) for 2 h at 4°C. Membrane protein was extracted using a Mem-PER Plus Membrane Protein Extraction Kit (89842, Thermo Fisher Scientific). Briefly, the cells were permeabilized with a mild detergent containing the permeabilization buffer, with the aim of liberating soluble cytosolic proteins. After removal of the soluble fraction containing hydrophilic proteins, the membrane proteins were extracted from the insoluble fraction using a membrane solubilization buffer.
Lysosome isolation kit
Manufactured by Abcam
The Lysosome isolation kit is a laboratory tool designed to isolate and purify lysosomes from cell samples. Lysosomes are subcellular organelles responsible for the breakdown and recycling of cellular components. The kit provides a standardized procedure for the efficient separation of lysosomes from other cellular fractions, enabling researchers to study the structure, function, and composition of these important organelles.
Automatically generated - may contain errors
Lab products found in correlation
Microcon 10kda centrifugal filter unit with ultracel 10 membrane, by Merck Group (2 mentions)
Mem per plus membrane protein extraction kit, by Thermo Fisher Scientific (1 mentions)
Clarity reagent, by Bio-Rad (1 mentions)
Criterion precast 4 20 gels, by Bio-Rad (1 mentions)
Lysosome extraction kit, by Merck Group (1 mentions)
Optima xe 100, by Beckman Coulter (1 mentions)
5 protocols using lysosome isolation kit
1
Lysosomal Membrane Protein Extraction
2
Retinal Protein Isolation and Analysis
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Retinas were harvested, flash frozen in liquid nitrogen, and later homogenized in 250 µL extraction buffer, as previously described (27 (link)). Lysosomes were purified using a Lysosome Isolation Kit (Abcam) according to the manufacturer’s instructions. Retinal homogenates, cell lysates, and protein isolates were fractionated using Criterion Precast 4–20% gels (Bio-Rad Laboratories). Proteins were transferred to polyvinylidene fluoride, blocked in 5% milk in Tris-buffered saline Tween 20, washed, and incubated overnight at 4°C with the appropriate antibodies (Supplementary Table 2 ). The antigen-antibody interaction was visualized with enhanced chemiluminescence Clarity Reagent (Bio-Rad Laboratories) using a ProteinSimple Fluorochem E.
3
Lysosomal Isolation and SARS-CoV-2 RNA Quantification
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For the lysosome isolation, 1 × 107 Huh-7 or MDCK cells were treated with 30 µM AKS466, and subsequently infected with SARS-CoV-2 or Influenzavirus A. The infections were performed in duplicates. After 24 h incubation, cells were trypsinized, and the duplicates were pooled. According to the manufacturer’s instructions, lysosome isolation was performed with a lysosome isolation kit (Abcam, Cambridge, Britain). In brief, the cell pellets were resuspended in a 1.5 mL isolation buffer, and homogenized with a precooled Dounce homogenizer. After centrifugation at 500× g for 10 min, 375 µL supernatant was layered on a 4 mL discontinuous density gradient, followed by centrifugation for 2 h by 145,000× g at 4 °C. For further purification, the lysosome fraction was diluted in 3.5 mL PBS, and lysosomes were pelleted by centrifugation for 1 h by 215,000× g at 4 °C. The lysosome pellet was resuspended in 100 µL PBS for 15 min. Viral RNAs of 30 µL of the lysosomal fraction were isolated and quantified by RTqPCR, as described above.
4
Isolation and Extraction of Lysosomal Fractions
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Lysosomes were isolated emplyoing a lysosome isolation kit (Abcam, #ab234047) following the manufacturer's protocol. Briefly, cells were collected into 15 mL conical tubes and isolated using an isolation buffer. The supernatant was centrifuged at 500 ×g for 10 min at 4°C and layered onto a discontinuous density gradient. Lysosomes were further isolated via ultracentrifugation (Beckman, Optima XE-100) at 145,000 g for 2 h at 4°C. Lysosomal fractions were extracted from cell homogenates using a lysosome extraction kit (Sigma-Aldrich; LYSISO1). Cell homogenates were centrifuged at 1000 × g for 10 min at 4°C. The supernatant fraction was centrifuged at 20,000 × g for 20 min at 4°C to pellet lysosomes and other organelles. The supernatant was collected as the cytosolic fraction. Pellet fractions were subjected to additional centrifugation. The ultimate pellet, lysosomal fraction, was lysed using the lysis buffer described within the procedure. Samples were then subjected to western blot analysis.
5
Lysosomal Fraction Isolation Protocol
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Supernatants of cells were concentrated using a Microcon-10kDa Centrifugal Filter Unit with Ultracel-10 membrane (Millipore, MRCPRT010). The lysosome isolation kit (Abcam, ab234047) was used to isolate lysosomal fractions from cultured cells by differential centrifugation followed by density gradient centrifugation. Finally, the purified lysosomal fraction was obtained using an ultracentrifuge (145,000 × g) for 2 h at 4°C.
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