| Atribut | Detail |
|---|---|
| Nomor kucing | SN0020 |
| Ukuran | 50T/100t |
| Penyimpanan | Stored at 4 °C for short storage. For longer storage, the kit can be stored at -20 °C or -80 °C. |
Komponen Perangkat
| Komponen | 50T | 100T |
| Lysis Buffer | 100ml | 200ml |
| Reagent A | 2.5ml | 5ml |
| Medium Buffer | 25ml | 50ml |
| Store Buffer | 5ml | 10ml |
Deskripsi Produk
- Isolates pure nuclei from animal cells and tissues (soft: liver/brain, hard: otot, cultured)
- Aplikasi: cell death (apoptosis), signaling, metabolism & protein studies
- Nuclei free from protein & nuclease contamination
Protokol
Persiapan Sampel:
- tisu:
- Take 100-200mg fresh tissue (liver, brain, heart)
- Wash with PBS/saline, kering, cut into small pieces
- Homogenize with 1ml pre-cooled Ly10is buffer & 50µL Reagent A (ice bath, 20X)
- Cultured Cells:
- Digest and wash cells
- Mesin sentrifugal (5-10menit, 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.
- Mesin sentrifugal (5menit, 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).
- Mesin sentrifugal (5menit, 700G, 4°C), discard supernatant (nuclear pellet remains).
- Resuspend nuclear pellet with 5ml Medium Buffer.
- Mesin sentrifugal (10menit, 1000G, 4°C), discard supernatant (purified nuclei pellet).
Penyimpanan:
- Resuspend purified nuclei in 50-100μl Store Buffer or desired buffer.
- Nuclei are ready for use or can be stored at -70°C
Catatan:
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.
- tisu: 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)
Aplikasi hilir:
-
- For Western Blot and 2D-PAGE, directly lyse the nuclear sample by adding loading buffer.
Produk-produk terkait
P1020 1×PBS, PH7.2-7.4, 0.01M
P1015 SDS-PAGE loading buffer,4×(dengan DTT)
SM0020 Mitochondrial Extraction Kit
Ulasan
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