Nuclear Extraction Protocol

互联网2013-11-13

1977

实验试剂

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)

Cell Extraction Buffer Formulation

100 mM Tris, pH 7.4
2 mM Na₃ VO₄
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 Na₄ P₂ O₇

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.

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% NP₄ 0) 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).

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Nuclear Extraction Protocol

实验试剂

实验设备

实验材料

实验步骤