| 屬性 | 細節 |
|---|---|
| Cat Number | SN0020 |
| 尺寸 | 50T/100T |
| 貯存 | Stored at 4 °C for short storage. For longer storage, the kit can be stored at -20 °C or -80 ℃. |
套件組件
| 成分 | 50時間 | 100時間 |
| Lysis Buffer | 100毫升 | 200毫升 |
| Reagent A | 2.5毫升 | 5毫升 |
| Medium Buffer | 25毫升 | 50毫升 |
| Store Buffer | 5毫升 | 10毫升 |
產品描述
- Isolates pure nuclei from animal cells and tissues (soft: liver/brain, hard: 肌肉, cultured)
- 應用領域: cell death (apoptosis), signaling, metabolism & protein studies
- Nuclei free from protein & nuclease contamination
Protocol
樣品製備:
- 紙巾:
- Take 100-200mg fresh tissue (liver, brain, heart)
- Wash with PBS/saline, 乾燥, cut into small pieces
- Homogenize with 1ml pre-cooled Ly10is buffer & 50µL Reagent A (ice bath, 20X)
- Cultured Cells:
- Digest and wash cells
- 離心機 (5-10分分鐘, 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.
- 離心機 (5分分鐘, 700G, 4℃), 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).
- 離心機 (5分分鐘, 700G, 4℃), discard supernatant (nuclear pellet remains).
- Resuspend nuclear pellet with 5ml Medium Buffer.
- 離心機 (10分分鐘, 1000G, 4℃), discard supernatant (purified nuclei pellet).
貯存:
- Resuspend purified nuclei in 50-100μl Store Buffer or desired buffer.
- Nuclei are ready for use or can be stored at -70°C
筆記:
Ensuring Complete Nuclei:
- Low Temperature: Perform the entire procedure at a low temperature.
- 速度: Work quickly throughout the process.
- Cell Disruption: The key is to break cells without damaging organelles.
- 紙巾: 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 (公分)
Downstream Applications:
- For Western Blot and 2D-PAGE, directly lyse the nuclear sample by adding loading buffer.
相關產品
P1020 1×PBS, PH7.2-7.4, 0.01中號
P1015 安全資料表-PAGE loading buffer,4×(with DTT)
SM0020 Mitochondrial Extraction Kit
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