REGISTRATION NUMBER: IT - 12470 NUREX UNI EN ISO 9001: 2000 CERT. N° 1416 _______________________________________________________________________ biotecnologie, servizi per la ricerca Global Cell Fractionation Kit For extraction of proteins Cod: GCF-40 For laboratory use. Not for drug, household or other use. Application The Global Cell Fractionation Kit has been developed for the preparation of nuclear, cytoplasmic and whole extracts from cells or tissue. Each kit provides reagents for 40 extractions from 6 x 107 cells, which correspond to HT29 cells grown to confluence in a T150 cells culture flask. Materials supplied Wash Buffer Lysis Buffer 1 Lysis Buffer 2 Protease Inhibitor Solution Detergent Solution 2x100 ml 1x100 ml 1x10 ml 1x 2 ml 1x 0.5ml Instructions for use Cell collection 1. Wash cells twice with 5 ml ice-cold PBS. 2. Add 5 ml ice-cold Wash Buffer (supplemented with 50 ul Protease Inhibitor Solution). 3. Remove cells from culture plate by gentle scraping with cell-scraper and transfer cells to a pre-chilled 15 ml conical tube. 4. Centrifuge cell suspension for 5 min at 450 x g in a centrifuge precooled to 4°C. Discard supernatant. Keep cell pellet on ice. Cell lysis and isolation of cell nuclei 5. Gently resuspend the cells in 1 ml Lysis Buffer 1 (supplemented with 10 ul Protease Inhibitor Solution and 0.1 M DTT) 6. Add 25 µl Detergent Solution to the cell suspension. 7. Incubate suspension for 8’ in ice 8. Centrifuge cell suspension for 5 min at 1,300 x g in a microcentrifuge pre-cooled to 4°C. 9. Transfer the supernatant (CP fraction) into a new microcentrifuge tube and store at –20°C. 10. Wash the pellet with 1 ml of Lysis Buffer 1 11. Resuspend the pellet (which contains cell nuclei) in 200 µl Lysis Buffer 2 (supplemented with Protease Inhibitor Solution and 0.1 M DTT) 12. Centrifuge the suspension of nuclei for 5 min at 1,700 x g in a precooled microcentrifuge at 4°C. 13. Transfer the supernatant NP fraction) into a new microcentrifuge tube and store at –20°C and save the pellet (CR fraction) Extraction of membrane proteins 14. Centrifuge CP fraction for 30min at 16,000 x g in a microcentrifuge pre-cooled to 4°C. 15. Separe supernatant containing citosol protein (CS fraction) from pellet containing membrane protein (MP fraction) _______________________________________________________________________ NUREX srl Z.I. Predda Niedda - Strada n°3 – complesso “I Docks”, 2°piano – 07100 SASSARI Sede Legale: Loc. Serra Li Lioni Str. Vic. S.Maria di lu Cardu, 14/a - 07100 SASSARI Tel: 079 2677073 Fax: 079 2677356 E-mail: [email protected] Cod.Fisc./P.IVA: 01689490900 REGISTRATION NUMBER: IT - 12470 NUREX UNI EN ISO 9001: 2000 CERT. N° 1416 _______________________________________________________________________ biotecnologie, servizi per la ricerca Storage and stability Wash Buffer Lysis Buffer 1 and 2and Protease Inhibitor Solution Protease Inhibitor Solution Detergent Solution +4°C +4°C up to six months, for prolonged storage aliquots the buffers and frezze them at -20°C. +4°C up to six months, for prolonged storage aliquots the buffers and frezze them at -20°C. +4°C +4°C Yield of protein from 6 x 10 6 cells Type Whole-cell extract Nuclear extract Cytoplasmatic extract Membrane extract Amount ~2.5 mg ~0.3 mg ~2.0 mg ~0.03 mg Co ncentration ~2.5 mg/ml ~1.5 mg/ml ~2.0 mg/ml ~5.0 mg/ml The kit procedure is a simple, fast and effective way to obtain proteins contained in cytoplasmic and nuclear compartments of the cell. Cytoplasmic proteins are obtained in two steps: • The cells are collected in ice-cold PBS in the presence of Protease Inhibitors to prevent proteolysis. • Addition of the Detergent causes leakage of the into the supernatant. Nuclear proteins are solubilized in the Lysis Buffer in the presence of the Protease Inhibitor Cocktail _______________________________________________________________________ NUREX srl Z.I. Predda Niedda - Strada n°3 – complesso “I Docks”, 2°piano – 07100 SASSARI Sede Legale: Loc. Serra Li Lioni Str. Vic. S.Maria di lu Cardu, 14/a - 07100 SASSARI Tel: 079 2677073 Fax: 079 2677356 E-mail: [email protected] Cod.Fisc./P.IVA: 01689490900
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