Nuclear Extraction Protocol
实验概要
The procedure presented below describes a method for extracting nuclear from several cell lines of human origin.
主要试剂
Hypotonic Buffer Solution
20 mM Tris-HCl, pH 7.4
10 mM NaCl
3 mM MgCl2
Phosphate Buffered Saline
NP40 Detergent (10%)
Cell Extraction Buffer (Cat. no. FNN0011)
PMSF (1 mM)
Protease Inhibitor Cocktail, Sigma (Cat. no. P-2714)
Quant-iT™ Protein Quantitation Kit (Cat. no. Q33210)
Or
Cell Extraction Buffer Formulation
100 mM Tris, pH 7.4
2 mM Na3VO4
100 mM NaCl
1% Triton X-100
1 mM EDTA
10% glycerol
1 mM EGTA
0.1% SDS
1 mM NaF
0.5% deoxycholate
20 mM Na4P2O7
This Cell Extraction Buffer may be apportioned into 1x aliquots in microcentrifuge tubes and stored at –20°C until ready for use. Thaw on ice prior to extracting cells.
The Cell Extraction Buffer must be supplemented with 1 mM PMSF (not included) and Protease Inhibitor Cocktail (not included) just prior to use to make Complete Cell Extraction Buffer. Addition of the Protease Inhibitor Cocktail and PMSF is necessary to inhibit proteolysis in cell extracts. For the PMSF addition, we recommend making a 0.3 M stock in DMSO, and adding sufficient volume for a final concentration of 1 mM (i.e., 17 μl per 5 ml Cell Extraction Buffer). PMSF is very unstable and must be added just prior to use, even if added previously. For the Protease Inhibitor Cocktail addition, we recommend Sigma (Cat. no. P-2714), reconstituted according to the manufacturer’s instructions, and adding 250 μl per 5 ml Cell Extraction Buffer. The stability of protease inhibitor-supplemented Cell Extraction Buffer is 24 hours at 4°C.
主要设备
Microfuge Tubes
实验材料
Cell lines of human origin
实验步骤
Researchers should optimize the cell extraction procedures for their own applications.
1. Collect cells (5 x 106) in PBS by centrifugation (non-adherent) or scraping from culture flasks (adherent).
2. Wash cells twice with cold PBS.
3. Remove and discard the supernatant and collect the cell pellet.
4. Transfer the cells into a prechilled microcentrifuge tube.
5. Gently resuspend cells in 500 μl 1x Hypotonic Buffer by pipetting up and down several times. Incubate on ice for 15 minutes.
6. Add 25 μl detergent (10% NP40) and vortex for 10 seconds at highest setting.
7. Centrifuge the homogenate for 10 minutes at 3,000 rpm at 4°C.
8. Transfer and save the supernatant. This supernatant contains the cytoplasmic fraction. The pellet is the nuclear fraction.
9. Resuspend nuclear pellet in 50 μl complete Cell Extraction Buffer for 30 minutes on ice with vortexing at 10 minute internals.
10. Centrifuge for 30 minutes at 14,000 x g at 4°C. Transfer supernatant (nuclear fraction) to a clean microcentrifuge tube.
11. Aliquot and store at –80°C. The nuclear extracts are ready for assay.
12. Quantitate protein concentration using the Quant-iT™ Protein Quantitation Kit (Cat. no. Q33210).
