Native cell nuclear extraction
Native tissue nuclear extraction (Manual)
Increased awareness of cellular proteins as potential drug targets or as biological markers for the detection of disease conditions necessitates the development of new protein sample preparation techniques from tissue or cells.
This nuclear extraction kit
covers 1) the extraction of nuclear proteins from whole
cells/tissues, 2) removal of interfering substances,
and 3) prevention of protein degradation, providing
nuclear proteins that are directly
compatible with downstream analytical applications.
Viagene Nuclear extraction kits provide reproducible, reliable and convenient protein sample preparation to achieve biologically relevant data by EMSA, immunoblotting, ELISA, array, activity assay, 2D gel electrophoresis and mass spectrometry.
B. Major procedure:
1. Touch cells - nuclear protein extraction:
1. aspirate medium
2. wash with ice-cold PBS
3. add 3 mL ice-cold PBS and scrape cells gently into 15mL tube
4. centrifuge 5 minutes at 500 rpm, 4OC
5. carefully aspirate supernatant with pipet
6. resuspend pellet in 1 mL ice-cold Working Sol. I
7. incubate 15 minutes on ice to allow cells to swell
8. add 100 ul of Solution II to 1 mL ice-cold Working Sol. I
9. vortex 10 seconds
10. centrifuge 2-3 minutes at maximum speed (~15K RPM)
11. carefully aspirate supernatant with pipet; this is the cytoplasmic fraction
12. resuspend the pellet in 175 ul ice cold Working Sol. III
13. vortex 30 seconds; rotate vigorously at 4OC 30 minutes
14. centrifuge 15 minutes at maximum speed; remove the supernatant to a fresh, chilled tube. leave on ice if using immediately, or store aliquots at –80OC until use. DO NOT freeze/thaw.
15. assay for protein concentration (Pierce’s BCA kit).
2. Suspension cells - nuclear
protein extraction: (see other section)
C. Pack (for 50 samples) list:
|5 X Buffer A||1 bottle||35 ml/bottle|
|2X Buffer B||1 vial||1.5 ml/vial|
|Solution I||1 vial||1.75 ml/vial|
|Solution II||1 vial||1.75 ml/vial|
|Solution III||1 vial||1.5 ml/vial|
|PMSF Solution||1 vial||0.85 ml/vial|
|User Manual||1 set||1|