| Attributo | Dettagli |
|---|---|
| Cat Number | SN0020 |
| Misurare | 50T/100T |
| Magazzinaggio | Stored at 4 °C for short storage. For longer storage, the kit can be stored at -20 °C or -80 °C. |
Componenti del kit
| Componenti | 50T | 100T |
| Lysis Buffer | 100ml | 200ml |
| Reagent A | 2.5ml | 5ml |
| Medium Buffer | 25ml | 50ml |
| Store Buffer | 5ml | 10ml |
Descrizione del prodotto
- Isolates pure nuclei from animal cells and tissues (soft: liver/brain, hard: muscolo, cultured)
- Applicazioni: cell death (apoptosis), signaling, metabolism & protein studies
- Nuclei free from protein & nuclease contamination
Protocollo
Preparazione del campione:
- Tessuti:
- Take 100-200mg fresh tissue (liver, brain, heart)
- Wash with PBS/saline, Asciutto, 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), scartare il surnatante, 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), scartare il surnatante (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), scartare il surnatante (nuclear pellet remains).
- Resuspend nuclear pellet with 5ml Medium Buffer.
- Centrifuga (10min, 1000G, 4°C), scartare il surnatante (purified nuclei pellet).
Magazzinaggio:
- Resuspend purified nuclei in 50-100μl Store Buffer or desired buffer.
- Nuclei are ready for use or can be stored at -70°C
Appunti:
Ensuring Complete Nuclei:
- Low Temperature: Perform the entire procedure at a low temperature.
- Velocità: Work quickly throughout the process.
- Cell Disruption: The key is to break cells without damaging organelles.
- Tessuti: 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.
prodotti correlati
P1020 1×PBS, PH7.2-7.4, 0.01M
P1015 Scheda di sicurezza-PAGE loading buffer,4×(con DTT)
SM0020 Mitochondrial Extraction Kit
Recensioni
Non ci sono ancora recensioni.