Mini protean tetra cell electrophoresis chamber

Protein pellets (isolated as described for the DNA extraction) were suspended in 5x pellet volume of RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5 % sodium deoxycholate, 0.1 % SDS, 1 % triton X-100) and incubated on ice for ~30 min. The lysate was spun for 10 min at 4oC, at 14,000 rpm and the supernatant was combined with 4x SDS gel-loading buffer (62.5 mM Tris-H₃PO₄, pH 7.5, 1 mM EDTA, 2 % SDS, 10 mM DTT, 1 mM NaN₃ and 33% glycerol) and fresh 15% β-mercaptoethanol. Samples were boiled at 100oC for 5 min and loaded on SDS-PAGE gradient gel (Biorad). Electrophoresis was performed using Mini-PROTEAN Tetra cell electrophoresis chamber (Qiagen) for 2 h at 120 V. The resolved proteins were transferred on Whatman nitrocellulose membrane using a trans-blot electrophoretic transfer system according to the manufacturer’s protocol, blocked with 5% milk PBST and probed with the antibodies against FKBP-12 (1:500), Cre (1:500) and enolase (1:1000) (all Abcam), followed by a compatible HRP-labelled secondary antibody. Enhanced chemiluminescence system (ECL) was used to visualise the proteins on X-ray film.