Patent ID: 6921817

Claim:
A method for simultaneous isolation of biologically active transcription factors and DNA, wherein no ultracentrifugation or sonication is used, comprising the following steps: a) collect cells from a culture or from a patient, b) washing said cells at least once with PBS, c) suspend and maintain said cells in buffer A (cell lysis buffer) for approximately 15 minutes wherein buffer A comprises: 20 mM Hepes, pH 7.9, 10 mM NaCl, 3 mM MgCl 2 , 0.1% NP-40, 10% glycerol, 0.2 mM EDTA, 1 mM DTT, 0.4 mM PMSF, 1 μg/ml antipain, 1 μg/ml leupeptin, d) centrifuge the suspension of step c at approximately 2,000 rpm for approximately 5 min at approximately 4° C., e) remove the upper cytoplasmic supernatant fraction and then clarify this fraction by adding buffer D (cytoplasmic extraction clarification buffer ) at approximately 4° C., wherein buffer D comprises: 20 mM Hepes, pH 7.9, 400 mM NaCl, 0.2 mM EDTA, 40% glycerol, 1 mM DTT, 0.4 mM PMSF, 1 μg/ml antipain, 1 μg/ml leupeptin, f) centrifuge the clarified fraction formed in step e approximately 13,000 rpm for approximately 15 min, g) remove and freeze the top clear supernatant on dry ice then store the frozen top clear supernatant at approximately −86° C., h) wash the bottom nuclear fraction formed at the end of step d with buffer B (extraction buffer without salt) wherein buffer B comprises: 20 mM Hepes, pH 7.9, 0.2 mM EDTA, 20% glycerol 1 mM DTT, 0.4 mM PMSF, 1 μg/ml antipain, 1 μg/ml leupeptin, i) centrifuge the mixture formed in step h at approximately 2,000 rpm for approximately 5 minutes at approximately 4° C., thereby pelleting cellular nuclei, j) suspend the pelleted nuclei of step i in buffer C (extraction buffer with salt) on ice, tapping the suspended mixture for approximately 45 minutes whereby nuclear proteins are extracted, wherein buffer C comprises: 20 mM Hepes, pH 7.9, 400 mM NaCl, 0.2 mM EDTA, 20% glycerol, 1 mM DTT, 0.4 mM PMSF, 1 μg/ml antipain, 1 μg/ml leupeptin, k) centrifuge the mixture formed in step j at approximately 13,000 rpm for approximately 15 minutes, at approximately 4° C., l) remove the top clear supernatant comprising biologically active transcription factors from the bottom nuclear fraction containing nucleic acids, and quick freeze on dry ice and store at approximately −86° C., m) extract DNA from said bottom nuclear fraction.