Ripa low lysis buffer

AN3 CA cells were lysed in RIPA (Low) lysis buffer (Meilunbio, China, Cat No: MA0153). For immunoprecipitation, the cell lysates were incubated with anti-FBXO7/IgG Protein A/G immunoprecipitated magnetic beads (Bimake, China, Cat No: B23201) for 4 h at 4 °C. The protein was eluted with SDS-PAGE loading buffer for 5 min at 100 °C. The results of the co-IP were detected by western blotting using the corresponding primary antibodies.

The HEK-293T cells were transfected with the indicated constructs. 36 h after transfection, the cells were treated with 20 μM MG132 for 8 h before harvesting. and then lysed in RIPA (Low) Lysis Buffer (Meilunbio, China, Cat No: MA0153). For immunoprecipitation, the cell lysates were incubated with anti-FLAG M2 agarose beads (Cat No. M8823) for 4 h at 4 °C. The bound beads were then washed four times with BC100 buffer (20 mM Tris-Cl, pH 7.9, 100 mM NaCl, 0.2 mM EDTA, 20% glycerol) containing 0.2% Triton X-100. The protein was eluted with FLAG peptide (Cat No. F4799) for 4 h at 4 °C. The results of the co-IP were detected by western blotting using the corresponding primary antibodies.

An epitope-tagging strategy to isolate INF2-containing protein complexes from human cells. Briefly, HEK-293T cells were transfected with pCMV-FLAG-INF2 constructs. Tagged INF2 protein levels were detected by western blot analysis. For purification, cells were lysed in RIPA (Low) Lysis Buffer (Meilunbio, China, Cat No: MA0153) containing fresh protease inhibitor (Bimake, China, Cat No: B14001) on ice for 2 h. The homogenate was centrifuged for 30 min at 12,000 rpm at 4 °C. Cleared lysates were filtered through 0.45 μm spin filters (Millipore, USA, Cat No. SLHV033RB), and immunoprecipitated using anti-FLAG M2 agarose beads (Cat No. M8823). The bound polypeptides were eluted with the FLAG peptide (Cat No. F4799). The final elutes from the beads containing FLAG peptide were resolved by SDS-PAGE for Coomassie Blue staining.