[ Home ] [ Protocols ] [ Innate Immunity ] [ Gastric Ca ]

Nuclear Extract Preparation

For preparation of extract from 1 150mm plate of cells

Nuclei Preparation

Harvest and wash cells with PBS in 15 ml polypro tube

Lyse cells by resuspending in 2.5 ml Buffer A

Centrifuge 10' @ 800 Xg at 4^oC; remove supernatant

Nuclear Extract:

Resuspend nuclei in 2 volumes Buffer C

Extract 30' at 4^oC on rocker

Centrifuge 30' at 4^oC in microfuge

Optional: Dialyze supernatant against Buffer D; Centrifuge 15' at 4^oC

Aliquot and freeze supernatant at -70

Buffer A:                                 For 10ml

10 mM HEPES pH 7.9           100 µl of 1M
1.5 mM MgCl₂                         15 µl of 1M
10 mM KCl                               100 µl of 1M
0.5 mM DTT                              10 µl of 0.5M
1 µg/ml leupeptin                      10 µl of 1 mg/ml
2 µg/ml aprotonin                      20 µl of 1 mg/ml
1 µg /ml pepstatin A                 10 µl of 1 mg/ml
0.5mM PMSF                             10 µl of 0.5 M
10 mM ß-glycerophosphate    100 µl of 1 M
1 mM Na vanadate                    100 µl of 0.1 M
0.1% Triton X-100                          1 ml of 1%
                                                  H₂0 8.5 ml

Buffer C:                                     For 2.5 ml

20 mM HEPES pH 7.9                 50 µl of 1M
25% glycerol                                  0.625 ml
0.42 M NaCl                                1.05 ml of 1 M
1.5 mM MgCl₂                       3.75 µl of 1 M
0.2 mM EDTA                        1 µl of 0.5 M
1 µg/ml leupeptin                        2.5 µl of 1 mg/ml
2 µg/ml aprotonin                        5 µl of 1 mg/ml
1 µg /ml pepstatin A                 2.5 µl of 1 mg/ml
1 mM Na vanadate                 25 µl of 0.1 M
0.5 mM PMSF                               1.25 µl of 0.5 M
0.5 mM DTT                                 1.25 µl of 0.5 M
10 mM ß-glycerophosphate        25 µl of 1 M
                            H₂O                      0.708 ml

Buffer D:                              For 200 ml

20 mM HEPES pH 7.9                     4 ml of 1 M
100mM KCl                                     20 ml of 1 M
12.5 mM MgCl₂                              2.5 ml of 1 M
0.1 mM EDTA                                   40 µl of 0.5 M
2 mM DTT                                        0.8 ml of 0.5 M
10 mM ß-glycerophosphate              2 ml of 1 M
1 mM Na Vanadate                            2 ml of 0.1 M
17% glycerol                                     34 ml
                                H₂O              134.7 ml

BACK