Tissue specimens are homogenized in modified RIPA buffer to obtain the soluble proteins, and centrifuged to clarify. The pellet was further extracted with a second buffer to obtain the less soluble protein fraction. The lysate solution may appear turbid at cold temperatures due to insolubility of buffer components. The solution should clear upon warming to room temperature.
Extraction 1: PBS, pH 7.4 1 ug/ml Aprotinin 1 mM NaF
Modified RIPA Buffer: 1 mM EDTA 1 ug/ml Pepstatin-A 0.1% SDS
0.25% Na deoxycholate 1 ug/ml Leupeptin 1 mM PMSF
1 mM Na3VO4
Extraction 2: PBS, pH 7.4, 5.0 M Urea, 2.0 M Thiourea, 50mM DTT, 0.1% SDS