Nuclear fractionation protocol

互联网2013-11-13

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实验试剂

1. Buffer A
10 mM HEPES, 1.5 mM MgCl₂ , 10 mM KCl, 0.5 mM DTT,
0.05% NP40 (or 0.05% igepal or tergitol) pH 7.9

    To prepare 250 ml stock of buffer A:
    HEPES: 1M = 238.3 g/L, therefore 10 mM = 0.59 g/250 ml
    MgCl₂ : 1M = 203.3 g/L, therefore 1.5 mM = 0.076 g/250 ml
    KCl: 1M = 74.5 g/L, therefore 10 mM = 0.187 g/250 ml
    DTT: 1M = 154.2 g/L, therefore 0.5 mM = 0.019 g/250 ml
    NP40 = 0.05%

2. Buffer B
5 mM HEPES, 1.5 mM MgCl₂ , 0.2 mM EDTA, 0.5 mM DTT,
26% glycerol (v/v), pH 7.9

To prepare 250 ml stock of buffer B:

    HEPES: 1M = 238.3 g/L, therefore 5mM = 0.295 g/250 ml
    MgCl₂ : 1M = 203.3 g/L, therefore 1.5mM = 0.076 g/250 ml
    EDTA: 1M = 372.2 g/L, therefore 0.2mM = 0.0186 g/250 ml
    DTT: 1M = 154.2 g/L, therefore 0.5mM = 0.019 g/250 ml
    26% Glycerol (v/v) = 65 ml

3. 4.6 M NaCl – 87.66 g/326 ml

实验步骤

1. Prepare 1 ml of buffer A with added cocktail of usual inhibitors from frozen stock and store on ice.

2. Add 500 μl of buffer a per large petri dish on ice and scrape thoroughly, leave on ice for 10 min.

3. Centrifuge at 4°C at 3000 rpm for 10 min.

4. Remove supernatant and keep it (this will contain everything except large plasma membrane pieces, DNA, nucleoli), extract out 10 μl for Bradford assay.

5. On ice resuspend pellet in 374 μl of buffer B and add 26 μl of 4.6 M NaCl to give 300 mM NaCl (high salt helps lyse membranes and forces DNA into solution).

6. Homogenize with 20 full strokes in Dounce or glass homogenizer on ice.

7. Leave on ice for 30 min.

8. Centrifuge at 24,000 g for 20 min at 4°C.

9. Aliquot supernatant, remove 10 μl for Protein Quantification (ab102536) and store at -70°C.

For your convenience, please consider some of our cell fractionation kits such as ab109718.

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