| Attribute | pojedinosti |
|---|---|
| Cat Number | SN0020 |
| Size | 50T/100T |
| Storage | Stored at 4 °C for short storage. For longer storage, the kit can be stored at -20 °C or -80 °C. |
Kit Components
| Components | 50T | 100T |
| Lysis Buffer | 100ml | 200ml |
| Reagent A | 2.5ml | 5ml |
| Medium Buffer | 25ml | 50ml |
| Store Buffer | 5ml | 10ml |
Product Description
- Isolates pure nuclei from animal cells and tissues (soft: liver/brain, hard: mišića, cultured)
- Prijave: cell death (apoptosis), signaling, metabolism & protein studies
- Nuclei free from protein & nuclease contamination
Protocol
Sample Preparation:
- Tissues:
- Take 100-200mg fresh tissue (liver, brain, heart)
- Wash with PBS/saline, dry, cut into small pieces
- Homogenize with 1ml pre-cooled Ly10is buffer & 50µL Reagent A (ice bath, 20x)
- Cultured Cells:
- Digest and wash cells
- Centrifuga (5-10min, 800g), discard supernatant, collect & count cells
- Resuspend 5×10^7 cells in 1ml pre-cooled Lysis Buffer
- Add 50µL Reagent A, homogenize (ice bath, 20-30x)
Nuclear Isolation:
- Transfer homogenate to a 1.5ml centrifuge tube.
- Centrifuga (5min, 700g, 4°C), discard supernatant (nuclear pellet remains).
- Resuspend nuclear pellet with 0.5ml pre-cooled Lysis Buffer.
- Transfer to a new tube containing 0.5ml Medium Buffer (layered carefully).
- Centrifuga (5min, 700g, 4°C), discard supernatant (nuclear pellet remains).
- Resuspend nuclear pellet with 5ml Medium Buffer.
- Centrifuga (10min, 1000g, 4°C), discard supernatant (purified nuclei pellet).
Storage:
- Resuspend purified nuclei in 50-100μl Store Buffer or desired buffer.
- Nuclei are ready for use or can be stored at -70°C
Notes:
Ensuring Complete Nuclei:
- Low Temperature: Perform the entire procedure at a low temperature.
- Speed: Work quickly throughout the process.
- Cell Disruption: The key is to break cells without damaging organelles.
- Tissues: Use a small-capacity glass homogenizer with a tight-fitting pestle for efficient homogenization.
- Cultured Cells: Breaking adherent cells is more challenging. Utilize the small homogenizer and tight pestle for optimal results.
Centrifugation:
- Calculate the correct centrifugal speed (RPM) based on the desired relative centrifugal force (RCF or g-force) using the formula:
- G = 1.11 x 10^-5 x R x (RPM)^2
- G: RCF (g)
- RPM: Rotations per Minute (squared)
- R: Rotor Radius (cm)
Downstream Applications:
- For Western Blot and 2D-PAGE, directly lyse the nuclear sample by adding loading buffer.
Related Products
P1020 1×PBS, PH7.2-7.4, 0.01M
P1015 SDS-PAGE loading buffer,4×(with DTT)
SM0020 Mitochondrial Extraction Kit
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