- Process 50 extractions of 107 cells each
- Less than 10% contamination between nuclear and cytoplasm fractions
- 2 hour processing time
- Produces ~1 mg with with 2 mg/ml non-denature cytoplasm protein and ~0.5 mg with 5 mg/ml non-denature nuclear protein
Mammalian cells or fresh tissue
- NE Reagents-I, -II, and -III
- Protease Inhibitor Cocktail
- Phosphatase Inhibitor Cocktail I
- Phosphatase Inhibitor Cocktail II
Other Materials Required
- 1X PBS Buffer
- Precision pipettes to deliver 1 µl to 1 ml volumes
- Adjustable 1-25 ml pipettes for reagent preparation
- Tissue homogenizer for tissue samples
- Refrigerated centrifuge with swing out rotor and refrigerated microcentrifuge
- Other lab consumables
- Prepare all reagents and samples as instructed in the manual.
- Add appropriate amount of 1X NE Reagent-I for sample amount
- Culture Media: Disperse Cells
Tissue Samples: Homogeize using dounce homogenizer or tissue grinder
- Incubate 15 minutes on ice
- Add appropriate amount of 1X NE Reagent-II for sample amount
- Incubate 2 minutes on ice
- Centrifuge tubes at 4°C, 14000 x g for 30 seconds in microcentrifuge.
- Transfer supernatant (cytoplasm fraction) into new tube. Store at -80°C
- Add appropriate amount of 1X NE Reagent-III for sample amount
- Vortex on highest setting for 10 seconds to suspend pelletes completly
- Incubate for 40 minutes on ice, vortexing for 10 seconds every ten minutes
- Centrifuge tubes at 4°C, 14000 x g for 10 minutes in microcentrifuge. Transfer supernatant (nuclear fraction) to a new tube and store at 0-80°
The NE-50 extraction buffer is compatible with the BCA assay if diluted at least 5-fold.
NE reagent-I, II, and III may be stored for up to 6 months at 2° to 8°C from the date of shipment. Protease Inhibitor and Phosphatase Set-I, Set-II should be stored at -20°C or -80°C (recommended at 80°C). Note: the kit can be used within one year if the whole kit is stored at -20°C. Avoid repeated freeze-thaw cycles.