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| Protocol |
WESTERN BLOT PROTOCOL PROTEIN LYSATES |
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| WESTERN LYSATE BUFFER 100 mM TRIS, pH 8.0 2M 1.5 ml 1% NP40 100% 0.3 ml 0.15 M NACL 5m 0.9 ml 2 mM EDTA 0.5m 0.12 ml ddH20 25.96 ml PMSF 17.4 mg/ml 17.2 ul TPCK 1.0 mg/ml 0.3 ml TLCK 1.0 mg/ml 0.3 ml Leupeptin 1.0 mg/ml 0.3 ml Aprotinin 1.0 mg/ml 0.3 ml 30.0 ml PROTEASE INHIBITORS STOCK SOLUTIONS. STORE -200C. PMSF 17.4 mg/ml (0.1 M) in 100 % ETOH TPCK 1.0 mg/ml in 100 % ETOH TLCK 1.0 mg/ml in 10 mM TRIS, pH 7.4 Leupeptin 1.0 mg/ml in 10 mM TRIS, pH 7.4 Aprotinin 1.0 mg/ml in 10 mM TRIS, pH 7.4 Note: lysate buffer can be stored for up to 1 week at 40C once unfrozen. |
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| WESTERN BLOT PROTOCOL PROTEIN LYSATES 1) Near confluent T150 flask of cells. 2) Wash in ice-cold PBS X3 total (if conf luent, X2 5 ml PBS wash, f ollowed by scrape in 5 mls, spin, wash Xl in PBS; if floaters, wash x3 in PBS). Transfer to 15 ml conical. 3 ) Add approx. 1 ml of lysate buf f er. Gently vortex to resuspend. Let sit on ice 10 minutes. Note, about 1 ml of lysate buffer to 0.3 ml of packed cell volume gives a good concentration of protein , A 2 to 3 ug/ul. 4) Transfer to Eppendorf. Spin 3 min at 40C. 5) Carefully remove supernatant. Can keep nuclear fraction. Store at -700C. Assay protein per Bradford. Can assay total protein in gel by Commassie Blue stain, or total protein on nitrocellulose filter by NHSBiotin stain (Biorad). |
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| WESTERN BLOT PROTOCOL PROTEIN LYSATES |
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